Aim of the thesis - VU-DARE Home - Vrije Universiteit Amsterdam

VRIJE UNIVERSITEIT
Molecular aspects of the histamine H1 receptor
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Exacte Wetenschappen
op dinsdag 1 juni 2010 om 11.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Kamonchanok Sansuk
geboren te Bangkok, Thailand

promotor: prof.dr. R. Leurs
copromotor: dr. R.A. Bakker

To my parents,
who brought me into this world,
and shaped me into who I am,

ISBN: 9789461080394
Cover:Tibetan Thangka “Purification of the mind”
Photoshop by Rodrigo Reijers
Printed at: Gildeprint B.V., Enschede, the Netherlands

Contents
Chapter 1 Introduction to the histamine H1 receptor
AIM of the thesis
Chapter 2 Expression and purification of GPCRs
Chapter 3 Expression of the histamine H1 and H4
receptor by in vitro cell free expression
system and their mass spectrometric
analysis
Chapter 4 Mass spectrometric analysis of the H1R:
glycosylation and binding of an
irreversible H1R antagonist
Chapter 5 A structural insight into the reorientation
of transmembrane domains 3 and 5
during H1R activation
Chapter 6 The role of isoleucine at position 6.45 in
the H1R
Chapter 7 General discussion
Summary
Samenvatting
References
Acknowledgements
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CHAPTER
Introduction to the histamine H1 receptor
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Chapter 1
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Introduction to the H1R
The histamine H1 receptor (H1R) belongs to the membrane protein family of G protein-coupled
receptors (GPCRs). GPCRs or also known as seven-transmembrane domain (7TM) receptors
represent the largest class of membrane protein in the human genome (Jacoby, Bouhelal et al. 2006;
Schlyer and Horuk 2006; Lefkowitz 2007; Lagerstrom and Schioth 2008). All members of this
membrane protein family, including the H1R, share a common membrane topology: an extracellular
N-terminus, an intracellular C-terminus and seven transmembrane (TM) helices, which are
connected by three intra-cellular loops (ICLs) and three extracellular loops (ECLs) (Figure 1)
(Jacoby, Bouhelal et al. 2006; Schlyer and Horuk 2006; Lefkowitz 2007; Lagerstrom and Schioth
2008).
Figure 1. GPCR structure overview. This figure is obtained from Nygaard (Nygaard, Frimurer et
al. 2009).
Serpentine model of a GPCR with the seven transmembrane (TM) helical segments (TM-I to TM-VII). The
highly conserved disulfide bridge from the end of TM-III to the middle of extracellular loop 2 (ECL-2). The
model also show a helix VIII at the C-terminus. Conserved residues are indicated with orange (conserved) and
red (highly conserved) symbols. The three main residues are R3.50 of TM-III, W6.48 of TM-VI and Y7.53 of
TM-VII. Their presumed interaction partners are indicated by dotted lines and arrows (Nygaard, Frimurer et
al. 2009).

Introduction to the histamine H1 receptor
The H1R is expressed ubiquitously in lung smooth muscle, vascular endothelium, gastro-intestinal
tract, heart, and brain. Histamine, which is an endogenous ligand of the H1R, is derived from
histidine and stored in basophils, mast cells, lymphocytes, neurons, and gastric enterochromaffin-
like cells, (Barger and Dale 1910; Thurmond, Gelfand et al. 2008). Through the H1R , histamine
exerts many actions related to allergic inflammation (e.g. local reddening, wheal and flare of skin,
nasal congestion, and bronchoconstriction) (Thurmond, Gelfand et al. 2008) and CNS effects (e.g.
reduction of locomotor activity, exploratory behavior, and cognitive functions) (Inoue, Yanai et al.
1996).
Signaling pathway of GPCRs and the H1R
Figure 2. GPCR signaling. This figure is modified from Siehler (Siehler 2008).
The GPCRs coupled to Gs proteins stimulate adenylyl cyclase (AC) causing an increase in intracellular cyclic
adenosine mono-phosphate (cAMP) levels, whereas the GPCRs coupled to Gi proteins inhibit the formation of
cAMP. cAMP regulates various cellular function, through the activation of protein kinases such as protein
kinase A (PKA). The GPCRs coupled to Gq protein activate phospholipase C-β (PLC-β), resulting in the
hydrolysis of phosphatidylinositol-4, 5-biphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and
diacylglycerol (DAG). DAG increase the activity of PKC, and IP3 triggers the release of Ca 2+ from intracellular
stores. The GPCRs coupled to G12 activates PLC-ε and the small GTPase Rho.
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Chapter 1
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H1R agonists
H1R antagonists
Figure 3. Chemical structures of various H1R ligands

Introduction to the histamine H1 receptor
Originally, GPCR signaling is thought to be dependent on association with a heterotrimeric G
protein. Interaction of an activated GPCR with a G protein catalyzes the exchange of guanosine
diphosphate (GDP) by guanosine triphosphate (GTP), enabling Gα- and βγ-subunits to interact with
a variety of downstream effectors (Siehler 2008). Signaling of the Gα subunit is dependent on the
Gα isoform as shown in Figure 2. All the four members of the Gαq family (Gαq, Gα11, Gα14, and
Gα15/16) stimulate phospholipase C-β (PLC-β) enzymes (Berridge 1993; Rhee 2001; Offermanns
2003). The βγ-subunits also activate PLC-β, although with significantly lower affinities than the Gαq
(Rhee 2001). PLC-β then catalyzes the hydrolysis of phosphatidylinositol diphosphate (PIP2) to
form inositol triphosphate (IP3) and diacylglycerol (DAG) (Berridge 1993; Rhee 2001). Activating of
protein kinase C (PKC) by DAG modulates the activity of many cellular substrates including the
transcription factors like NF-κB (Berridge 1993; Marinissen and Gutkind 2001). IP3 on the other
hand, binds to IP3 receptors at the membrane of endoplasmic reticulum and causes the release of
Ca 2+ into the cytosol (Siehler 2008). The more recently described Gα12 stimulates phospholipase C-ε
(PLC-ε) causing increases of intracellular IP3 and DAG (Kelley, Kaproth-Joslin et al. 2006). In
addition to the activation of PLC-ε, Gα12 can also activate PKC through the small GTPase Rho (Sah,
Seasholtz et al. 2000; Siehler 2008). Gαs and Gαi proteins mostly modulate adenylyl cyclase (AC)
activity (Birnbaumer and Birnbaumer 1995; Offermanns 2003). The activation of AC results in the
formation of cAMP, which regulates other proteins such as protein kinase A (PKA) (Siehler 2008).
However, recent studies (Daaka, Luttrell et al. 1997; Vanhauwe, Thomas et al. 2002; Marinissen,
Servitja et al. 2003; Defea 2008) suggest that GPCR signaling is far more diverse than originally
thought. It is now generally accepted that GPCRs can couple to multiple G proteins (Daaka, Luttrell
et al. 1997). For example, the β2AR can couple to both Gαs and Gαi (Daaka, Luttrell et al. 1997) and
the protease-activated receptor-1 (PAR-1) can couple to Gαi/o, Gαq and Gα12/13 (Vanhauwe, Thomas
et al. 2002; Marinissen, Servitja et al. 2003). These complicated couplings of GPCRs demonstrate the
importance of evaluating multiple signaling pathways as one assay for the quantification of
receptor activation may not be sufficient. An increase in G protein coupling in response to agonist
binding can still be evaluated by quantifying membrane-associated GTPase activity. However, over
the last few years it has been shown that GPCRs can even signal without coupling to a G protein (see
review by (Defea 2008)). This G protein independent signaling is mediated through β-arrestins and
G-protein coupled receptor kinases (GRK) and can result in the activation of cytoplasmic effectors
such as Src, Ras and extracellular signal-regulated kinase ERK (Lefkowitz and Shenoy 2005; Reiter
and Lefkowitz 2006; Defea 2008).
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Chapter 1
The H1R mediates its action through the Gαq/11 protein (Leopoldt, Harteneck et al. 1997). As
described previously, the Gαq/11 protein activates PLC, which then hydrolyses PIP2 into IP3 and DAG.
Subsequently, DAG activates PKC, which in turn can phosphorylate other proteins, including the
H1R, resulting in receptor desensitization. IP3 causes the release of Ca 2+ from the intracellular store.
The elevation of intracellular Ca 2+ concentration can explain a variety of pharmacological
responses; for example the induction of smooth muscle cell contraction, the activation of nitric
oxide synthase (Satoh and Inui 1984), and the stimulation of phospholipase A2 (PLA2) (Murayama,
Kajiyama et al. 1990). Besides the Gαq/11 protein, the H1R can signal via the Gαi/o protein (Seifert,
Grunbaum et al. 1994) and is also reported to activate the transcription factor NF-κB (Leurs,
Church et al. 2002) either via βγ subunits in constitutively activate H1R or via both Gαq/11 and βγ
subunits (Bakker, Schoonus et al. 2001).
Figure 4. Interaction of retinal and a rotamer toggle switch of rhodopsin. This figure is
modified from Bhattacharya (Bhattacharya, Hall et al. 2008).
(A) Chemical structure of 11-cis-retinal and all-trans-retinal
(B) In the inactive state, the aromatic moiety of tryptophan W265 (W6.48) is parallel to the lipid bilayer
(Left). The indole nitrogen of the W6.48 also forms a water mediated hydrogen bond with N302 (N7.49) of the
conserved NPxxY motif of TM7. In the active state, the hydrogen bond with TM7 is disrupted when the
flipping of the W6.48 is triggered by the isomerization of 11-cis-retinal to all-trans-retinal.
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Introduction to the histamine H1 receptor
Figure 5. Crystal structures of Rhodopsin, β2AR and A2A adenosine receptor. This figure is
obtained from Hanson (Hanson and Stevens 2009).
The ECL-2 region (Top): The extracellular domain in rhodopsin serves to occlude the retinal binding pocket,
whereas in both the β2AR and A2A adenosine receptors the extracellular domain is highly constrained and
held away from the ligand binding pocket.
The binding pocket region (middle): The retinal binding pocket relies on hydrophobic interactions in addition
to a covalent linkage with lysine residue in TM5. The tail of carazolol, the β2AR ligand, forms polar
interactions with a conserved glutamate on TM3 and asparagine on TM7, whereas the head of carazolol forms
polar interactions with serine residues on TM5. The adenosine ligand ZM241385 forms polar interactions
with asparagine residues on TM6 and a glutamate on ECL-2
The intracellular interactions (Bottom): bovine rhodopsin is the only receptor with an intact ionic lock
interaction between the E/DRY motif and glutamate on TM6. All the other two structures have a disrupted
ionic lock. Thee E/DRY motif of β2AR and A2A adenosine receptor interacts with ICL-2 through a polar
interaction between the aspartate residue and either a serine or tyrosine residue on ICL-2.
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Chapter 1
Furthermore, GPCRs, including the H1R, can also activate their G protein in the absence of their
ligands. This basal activity is so called “constitutive activity” (Jacoby, Bouhelal et al. 2006; Kobilka
and Deupi 2007; Lefkowitz 2007). Many GPCRs, which undergo spontaneous mutations and posses
constitutive activity, can actually cause a variety of human illnesses (Spiegel 1998; Jacoby, Bouhelal
et al. 2006; Lefkowitz 2007). For example, the hypercalcemia and skeletal dysplasia in Jansen’s
disease is the result of a constitutively active parathyroid hormone (PTH) receptor (Schipani, Kruse
et al. 1995), the familial male precocious puberty is caused by a single point mutation in the
luteinizing hormone (LH) receptor (Shenker, Laue et al. 1993) and the congenital night blindness is
due to mutations in rhodopsin (Dryja, Berson et al. 1993). The discovery of constitutive activity has
also led to a reclassification of ligands. Full agonists are capable of maximal receptor stimulation.
Partial agonists are unable to give full activity. Neutral antagonists have no effect on signaling
activity (but can prevent other ligands from binding to a receptor). Full and partial inverse agonists
can reduce the level of basal or constitutive activity (Bond, Leff et al. 1995; Leurs, Church et al.
2002; Strange 2002; Kobilka and Deupi 2007).
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The H1R ligands
Since the discovery of the H1R, numerous histamine derivatives have been synthesized to enhance
agonist potency and selectivity. At first, the imidazole ring of histamine (Figure 3) was replaced by
other heterocyclic rings. The most prominent compounds found in this class are 2-(thiazol-2-yl)-
ethylamine and 2-pyridylethylamine (Donaldson and Hill 1986). Another strategy which focused on
shortening or elongation of the ethylamine side-chain, however, resulted in loss of potency (Zingel,
Leschke et al. 1995). The introduction of different substitutes at the C-2 position of the imidazole
ring resulted in selective H1R agonists, such as 2-(3-bromophenyl)histamine and 2-(3-
trifluoromethyl)-phenyl-histamine (Leschke, Elz et al. 1995). Interestingly, the addition of a
diphenylalkyl group, which is typically found in H1R antagonists, also lead to a more selective and
potent H1R agonist, called histaprodifen (Elz, Kramer et al. 2000). Another interesting ligand is 8R-
lisuride, which is a potent H1R agonist that is derived from ergot alkaloids. This compound is,
however, not selective since it is also known to bind dopamine and serotonin receptors. All these
H1R agonists may serve as valuable tools for the investigation of H1R mediated physiological and
pathological functions (Bakker, Weiner et al. 1994). The first generation antihistamines generally
contain the ethylamine moiety linked to two aromatic rings (Figure 3) such as mepyramine,
chlorpheniramine, hydroxyzine and diphenhydramine. However, fusion of the tricyclic rings is also

Introduction to the histamine H1 receptor
tolerated (e.g. doxepin and ketotifen). The first generation antihistamines were effective through
their strong histamine inhibiting activity. However, they also induce sedation and psychomotor
retardation among allergic patients, due to their ability to cross the blood brain barriers and bind to
muscarinic receptors or H1R in the central nervous system (Thurmond, Gelfand et al. 2008). The
second generation H1R antagonists were therefore developed with strongly reduced side effects.
Several of the second generation antihistamines such as cetirizine, loratadine and fexofenadine are
in use for the treatment of allergic symptoms.
Inactive-state GPCRs
Structural knowledge of GPCRs and the H1R
The first insights into the structure of GPCRs came from the crystal of bovine rhodopsin in year
2000 (See further details on protein expression at chapter 2) (Palczewski, Kumasaka et al. 2000).
Rhodopsin is well suited for structural studies due to its high expression level in nature (bovine
retina) and high stability compared to other GPCRs (Nygaard, Frimurer et al. 2009). Rhodopsin
consists of the 7TM protein “opsin” and the chromophore “11-cis-retinal” (Figure 4A), which is
covalently bound to the lysine residue K7.43 in TM7. The β-ionone ring (at the other end of the 11-
cis-retinal) is buried deeply in a hydrophobic pocket and has a direct interaction with the
tryptophan W6.48 (Palczewski, Kumasaka et al. 2000; Nygaard, Frimurer et al. 2009) from the
CWxP motif in TM6. This tryptophan is a part of important intra-molecular interaction known as a
“rotamer toggle switch”, which consists of residues F6.52, C6.47 and W6.48 (Nygaard, Frimurer et
al. 2009). Upon exposure to light, 11-cis-retinal isomerizs to all-trans-retinal, which induces a series
of conformational changes in the receptor, including a switching of the rotamer conformation of the
W6.48 into its active form (Figure 4B) (Bhattacharya, Hall et al. 2008; Nygaard, Frimurer et al.
2009). With the information obtained from the crystal structure of rhodopsin (Palczewski,
Kumasaka et al. 2000), earlier models of GPCRs based on the crystal structure of
bacteriorhodopsins (Luecke, Schobert et al. 1999) have been re-evaluated. In analogy to the
lipophillic binding pocket of 11-cis- retinal in the crystal structure of rhodopsin (Palczewski,
Kumasaka et al. 2000), the main ligand binding site of small molecule hormones and non-peptides
is thought to locate within the central crevice of the 7TM domains (Smit, Vischer et al. 2007;
Bhattacharya, Hall et al. 2008; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009).
Furthermore, the crystal structure of rhodopsin also revealed the proline kinks of TM1, 4 and 7
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Chapter 1
(Menon, Han et al. 2001), the bending of TM2 by a pair of G2.56 and G2.57 (Stenkamp, Filipek et al.
2002), the short eighth helix (Palczewski, Kumasaka et al. 2000) and the β-sheet at the ECL2 which
forms a lid together with N-terminus on top of the chromophore binding (Bourne and Meng 2000).
Figure 6. The superimposition of selected GPCR structures. This figure is obtained from Topiol
(Topiol and Sabio 2009).
(A) View from the toggle switch region (W6.48) of three GPCRs: the bovine rhodopsin with retinal (in
brown), the human β2AR with carazolol (in pink) and the human A2A adenosine receptor with ZM241385 (in
green). The residues corresponding to phenylalanine (F290) in the human β2AR is alanine (A) in the bovine
rhodopsin and histidine (H) in the human A2A adenosine receptor.
(B) View from the intracellular side of bovine rhodopsin and opsin with bound GαCT. TM6 (VI) of opsin
(yellow) is tilted outward by 6-7 Å. The glutamic residue E247 decouples from the ionic lock interaction with
R135 (R3.50) from the highly conserved DRY motif of TM3, allowing TM6 to move out and away from TM3
(III) and swing over to TM5 (V). This E247, then forms a salt bridge with K231 of TM5, helping TM6 to come
closer to TM5. The Y306 (Y7.53) from the NPxxY motif of TM7 is rotated into the helix core, blocking the
inward return of TM6.
The rhodopsin crystals stayed the only GPCR structure resolved to an atomic level for another
seven years until the crystal structures of human β2 adrenergic receptor (β2AR) (Cherezov,
Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007), the turkey β1 adrenergic receptor (β1AR)
(Warne, Serrano-Vega et al. 2008) and human A2A adenosine receptors (Jaakola, Griffith et al. 2008)
were resolved with the use of a variety of different protein-modification and engineering
approaches (see further details on protein expression at chapter 2). It has been shown that the four
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Introduction to the histamine H1 receptor
inactive state GPCR structures are overall in close agreement. The most significant structural
differences lie in the extracellular, intracellular and ligand-binding regions. It is known from the
rhodopsin crystals that the ECL2 region appears to form a β-sheet structure on top of the
chromophore binding cavity (Figure 5, ECL-2) (Palczewski, Kumasaka et al. 2000) and maybe act
as a lid shielding 11-cis-retinal from hydrolysis (Audet and Bouvier 2008; Hanson, Cherezov et al.
2008; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). However, in the β2-AR and β1AR X-
ray structures, the ECL2 loop is a short α-helix with cysteine bridges (Cherezov, Rosenbaum et al.
2007; Rasmussen, Choi et al. 2007; Warne, Serrano-Vega et al. 2008). It has been hypothesized that
the structural features of ECL2 region in β2AR and β1AR are more accessible to the extracellular
environment and allow the diffusion of ligands in and out of the binding pocket (Hanson, Cherezov
et al. 2008; Hanson and Stevens 2009; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009).
The ECL2 region of the A2A receptor, however, resembles neither the β-sheet of rhodopsin nor the
short α-helix of adrenergic receptors. In the region, the receptor adopts a random coil conformation
and has multiple cysteine bridges, in which three of them are linked to the ECL1 and one is linked to
the ECL3 (Jaakola, Griffith et al. 2008). This structural feature results in an open structure at the
extracellular end that can accommodate the entry of ligands (Hanson, Cherezov et al. 2008; Hanson
and Stevens 2009; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). Additional
structures will be needed to confirm whether the open binding pocket is a general feature of the
ligand-activated GPCRs.
Based on the crystal structures of rhodopsin, adrenergic and adenosine receptors; ligand binding is
mediated by polar and hydrophobic contact residues from TM3, 5, 6 and 7 (Cherezov, Rosenbaum
et al. 2007; Rasmussen, Choi et al. 2007; Jaakola, Griffith et al. 2008; Warne, Serrano-Vega et al.
2008). In rhodopsin (Figure 5, Binding pocket), 11-cis-retinal is located deep in the pocket with
its β-ionone ring reaching down between the indole side-chain of W6.48 (Palczewski, Kumasaka et
al. 2000). The adrenergic antagonists bind to their receptors similar to that of 11-cis retinal, but
they do not reach as deep into the pocket as retinal does. With regard to the toggle switch,
carazolol, a β2AR antagonist, interacts indirectly with the W6.48 via the phenylalanine F290
(Figure 6A) and force the tryptophan residue to be in its inactive conformation (Topiol and Sabio
2009). In contrast to the adrenergic and rhodopsin structures, ZM241385, an adenosine A2A
receptor antagonist, binds vertically to the bilayer plane of the A2A receptor and further away from
the W6.48. However, the receptor is still held in the inactive conformation via recruitment of H250
as an intermediate residue (Figure 6A) (Topiol and Sabio 2009). The observed differences between
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Chapter 1
the crystal structures of adrenergic and adenosine receptors suggest that the ligand-activated
GPCRs may have a wide variety of ligand binding modes (Hanson and Stevens 2009; Nygaard,
Frimurer et al. 2009; Topiol and Sabio 2009).
At the cytoplasmic surface, a major difference between the ligand-activated GPCRs and rhodopsin
lies in either the presence or absence of the polar interaction networks (also known as “ionic lock”)
between the highly conserved E/DRY motif on TM3 and a glutamate residue on TM6 (Hanson and
Stevens 2009; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). This ionic lock is
conserved among all the rhodopsin crystal structures and is thought to stabilize the inactive-state
conformation (Ballesteros, Jensen et al. 2001; Kobilka and Deupi 2007; Audet and Bouvier 2008;
Hanson and Stevens 2009; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). The ionic
lock has been observed in the inactive state rhodopsin crystals (Palczewski, Kumasaka et al. 2000),
but not in all the ligand-activated GPCRs structures (Cherezov, Rosenbaum et al. 2007; Rasmussen,
Choi et al. 2007; Jaakola, Griffith et al. 2008; Warne, Serrano-Vega et al. 2008). This can be
interpreted that either this interaction is so weak that it is broken during crystallization or it does
not exist in the ligand-bound states (Hanson and Stevens 2009; Nygaard, Frimurer et al. 2009;
Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). Interestingly, among the notable
differences found between the β1AR and β2AR structures are the residues in the ionic lock region. In
the β1AR crystal, a short α-helix is formed in the ICL2 region, and thereby accommodating a
hydrogen bond between Y149 on ICL2 and the aspartate of the E/DRY motif in TM3 (Warne,
Serrano-Vega et al. 2008). This structural feature is also observed in the A2A receptor crystal
(Jaakola, Griffith et al. 2008), but not in the β2AR and rhodopsin crystals (Palczewski, Kumasaka et
al. 2000; Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007) (Figure 5, bottom). The
absence of a short α-helix and the hydrogen bond network in the β2AR structure can be used to
explain the higher constitutive activity of the β2AR compared to that of the β1AR (Nygaard,
Frimurer et al. 2009; Rosenbaum, Rasmussen et al. 2009).
Active-state GPCRs
The activation of the rhodopsin family is thought to involve the disruption of intra-molecular
interactions that stabilize their inactive conformations (Smit, Vischer et al. 2007; Bhattacharya, Hall
et al. 2008; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). Based on homology
modeling (Bhattacharya, Hall et al. 2008) and biophysical experiments with several GPCRs
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Introduction to the histamine H1 receptor
(Dunham and Farrens 1999; Struthers, Yu et al. 2000; Altenbach, Klein-Seetharaman et al. 2001;
Huang, Li et al. 2001; Huang, Visiers et al. 2002; Shi, Liapakis et al. 2002), such as site-directed
mutagenesis (Huang, Li et al. 2001; Huang, Visiers et al. 2002), cysteine accessibility (Shi, Liapakis
et al. 2002), disulfide cross-linking (Struthers, Yu et al. 2000), spin labeling (Altenbach, Klein-
Seetharaman et al. 2001) and circular dichroism (Dunham and Farrens 1999), the cytoplasmic ends
of TM3 and TM6 are proposed to move away from one another during the activation process due to
the breaking of the ionic lock between the highly conserved E/DRY motif on TM3 and a glutamate
residue on TM6. The R3.50 is also restrained in the inactive conformation by a hydrophobic
arginine cage formed by conserved amino acids at position 3.46 (L: 15%, V: 18%, I: 58%, M: 15%)
and 6.37 (L: 37%, V: 24%, I: 20%, M: 5%) (Smit, Vischer et al. 2007). The breaking of the ionic lock
often results in constitutive activation of several GPCRs such as rhodopsin (Kim, Altenbach et al.
1997), H2R (Alewijnse, Timmerman et al. 2000), α1B adrenergic receptor (Scheer, Fanelli et al.
1996), luteinizing hormone receptor (Laue, Chan et al. 1995) and thyrotropin receptor
(Ringkananont, Van Durme et al. 2006). Another important intra-molecular interaction known as a
“rotamer toggle switch”, which consists of residues F6.52, C6.47 and W6.48 has also been proposed
to be involved in the activation process of the rhodopsin family (Smit, Vischer et al. 2007;
Bhattacharya, Hall et al. 2008; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio 2009). In the
inactive conformation, the plane of the aromatic moiety of W6.48 is parallel to the lipid bilayer,
while in the predicted active state, it is perpendicular to the lipid bilayer (Figure 4B)
(Bhattacharya, Hall et al. 2008). This rotamer flipping hypothesis is supported by the information
from the crystal structure of Meta I (the first stable intermediate of photo-activated rhodopsin) that
showed the toggling of the tryptophan rotamer (Ruprecht, Mielke et al. 2004). The toggling of
W6.48 is thought to modulate the bend angle around the highly conserved proline P6.50 on TM6
and leading to a movement of the cytoplasmic end of TM6 during the activation process (Shi,
Liapakis et al. 2002; Vilardaga 2006). In the inactive state, the highly conserved N7.49 of the NPxxY
motif in TM7 is restrained toward TM6 via water molecule in many rhodopsin family GPCRs (Smit,
Vischer et al. 2007). This asparagine residue is located in a hydrophobic cage formed by conserved
amino acids at position 2.46 (L: 91%) and 6.40 (L: 14%, V: 42%, I: 28%, M: 5%) (Smit, Vischer et al.
2007). It was proposed that upon receptor activation, N7.49 adopts the trans (t) conformation to
interact with D2.50 of the (N/S)LxxxD motif in TM2 (Urizar, Claeysen et al. 2005), and could force
the relocation of R3.50 from the ionic lock (Bakker, Jongejan et al. 2008). Mutations of asparagine at
position 7.49 or removal of the bulky and hydrophobic cage affects the constitutive activity of
several GPCRs such as rhodopsin (Han, Lin et al. 1996; Madabushi, Gross et al. 2004), thyroid
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Chapter 1
stimulating hormone receptor (Urizar, Claeysen et al. 2005), serotonin 5HT2A receptor (Shapiro,
Kristiansen et al. 2002) and H1R (Bakker, Jongejan et al. 2008). Shortly, it is suggested that a
mechanism for receptor activation may involve a separation of the TM3 and TM6, together with a
twist in TM6 which pulls the ICL3 into the cell and uncovers residues related to G-protein coupling
(Rovati, Capra et al. 2007; Audet and Bouvier 2008). This hypothesis is further supported by the
current crystal structure of opsin (Park, Scheerer et al. 2008). Opsin is the retinal-free
photoreceptor generated after the photo-activation and Schiff base hydrolysis of rhodopsin. There
are several subtle changes in the conformations of binding pocket residues, relative to rhodopsin:
the side chain of W6.48 moves into space previously occupied by 11-cis retinal, the interaction
between K7.43 and E3.28 is broken, the cytoplasmic end of TM6 is shifted outwards from the centre
of the TM bundle and became closer to TM5, the ionic lock is broken and Y7.53 from the NPxxY
motif on TM7 undergoes a conformational change and inserts into space occupied by TM6 in the
inactive state rhodopsin (Figure 6B). All these changes from inactive rhodopsin to active-state
opsin result in the creation of a cavity between TM3, TM5 and TM6, in which the G protein can bind
(Topiol and Sabio 2009).
Inactive-state H1R
Similar to other members of GPCRs, with the exception of rhodopsin, adrenergic receptors and
adenosine receptor, structural analysis at atomic level of the H1R are still limited by low expression
levels in both nature and heterologous expression systems and/or by difficulties in obtaining pure
and homogenous protein for crystallization. To date, the crystal structure of H1R has not been
solved yet. As a consequence, most of the structural studies of the H1R were only possible via
mutational analysis and computational techniques based on homology modeling with available
GPCR crystals. Based on various site-directed mutational analysis and homology modeling studies
(Leurs, Smit et al. 1994; Ohta, Hayashi et al. 1994; Moguilevsky, Varsalona et al. 1995; ter Laak,
Timmerman et al. 1995; Wieland, Laak et al. 1999; Jongejan, Bruysters et al. 2005; Jongejan and
Leurs 2005), the endogenous ligand histamine is believed to bind various amino acid residues in
TM3, TM5 and TM6 of the H1R (Figure 8). The H1R has a well conserved aspartate residue D3.32 in
TM3 that is believed to be one of the most crucial amino acids for ligand binding among biogenic
amine receptors. The residue D3.32 of the H1R is thought to form an ionic bond with the amino
group of histamine (Ohta, Hayashi et al. 1994; Jongejan and Leurs 2005). Several studies further
demonstrated that the asparagine N5.46 of the H1R is involved in ligand binding pocket by forming
20 | P a g e

Introduction to the histamine H1 receptor
an interaction with the distal nitrogen position (N τ ) of the imidazole ring of histamine (Leurs, Smit
et al. 1994; Moguilevsky, Varsalona et al. 1995; Jongejan and Leurs 2005), while the lysine residue
K5.39 interacts with the ortho nitrogen (N π ) of the imidazole ring of histamine (Leurs, Smit et al.
1995; ter Laak, Timmerman et al. 1995; Gillard, Van Der Perren et al. 2002; Jongejan and Leurs
2005). Mutation of residue K5.39 in guinea pig (Leurs, Smit et al. 1995), or in human (Gillard, Van
Der Perren et al. 2002) H1R resulted in a decrease in histamine potency and binding affinity.
Bruysters and co-workers also demonstrated that the serine S3.36 plays a part in the H1R binding
pocket and acts as a toggle switch in receptor activation mechanism (Jongejan, Bruysters et al.
2005). Furthermore, the residue F6.55 may be important in stabilizing the imidazole ring of
histamine, since mutation of F6.55 to alanine in both guinea pig and human resulted in a reduction
of histamine potency (Wieland, Laak et al. 1999). In short, amino acid residues, that are involved in
the histamine binding pocket consist of D3.32, S3.36 of TM3; K5.39, N5.46 of TM5 and F6.55 of
TM6. Bruysters and co-workers (Bruysters, Pertz et al. 2004) found that the residues D3.32 and
F6.52 are also crucial for the binding of histaprodifen, a selective H1R agonist that posses the
structural features of both agonist and antagonist. However, the binding of histaprodifen was not
affected by the mutation at residues K5.39 and F6.55. Furthermore, the mutation of N5.46 does not
influence the affinity of histaprodifen, although the signaling as measured by reporter gene assay is
greatly impaired. Interestingly, Bruysters and co-workers (Bruysters, Jongejan et al. 2005) also
observed the species selectivity of histaprodifen upon employing the mutant H1R, in which “human”
amino acids were individually replaced by their guinea pig counterparts. Mutation of N2.61 to
serine resulted in a complete reversal of human to guinea pig pharmacology. The conserved Y7.43
is also found to be pivotal in the mechanism behind the species selectivity. Based on these data, it
can be concluded that not all H1R agonists bind in a similar manner. The binding pocket for H1R
agonist may not be limited to only TM3, 5 and 6, but may also include TM2 and 7, as shown in
Figure 8 (Jongejan and Leurs 2005).
For antagonist binding, the residue D3.32 also appears to be an important interaction point.
Mutation of D3.32 to alanine resulted in a complete loss of [ 3H]mepyramine binding affinity (Ohta,
Hayashi et al. 1994; Nonaka, Otaki et al. 1998). However, the radioligand [ 3H]KW-4679 was shown
to bind to the D3.32A H1R mutant with a reasonable affinity compared to that of the wild type H1R
(D3.32A: KD = 49 nM and wild type: KD = 2.5 nM), showing that the tricyclic H1R antagonists such as
KW-4679 may have different binding pocket than mepyramine (Nonaka, Otaki et al. 1998).
Mutation of N5.46 to alanine resulted in an unaltered affinity of [ 3H]mepyramine,
21 | P a g e

Chapter 1
chlorpheniramine, cetirizine, and loratadine, suggesting that histamine and its antagonists may
interact with different amino acids in the same binding pocket (Leurs, Smit et al. 1994; Ohta,
Hayashi et al. 1994; Moguilevsky, Varsalona et al. 1995).
Figure 8. Binding of H1R ligands to the H1R. The figure is obtained from Jongejan (Jongejan and
Leurs 2005).
(A) The H1R model was obtained using the rhodopsin template. The amino group of histamine form an
interaction with D3.32 of TM3. The distal nitrogen position (N τ ) of the imidazole ring of histamine forms an
interaction with N5.46 of TM 5. The ortho nitrogen (N π ) of the imidazole ring of histamine forms an
interaction with K5.39 of TM5.
(B) The classical binding site (light gray) showing residues involved in binding of classical H1R agonists:
D3.32, K5.39, T5.42, N5.46, F6.52 and F6.55. The binding pocket of histaprodifen (darker grey) extends
from the classical pocket to TM2 and TM7.
The introduction of a carboxylic moiety in many second generation H1R antagonists showed to limit
brain penetration, thus reducing sedative side effects. Despite the beneficial pharmacokinetic
properties, this carboxylic moiety also seems to affect the binding affinity. Mutation of the guinea
pig H1R at the positively charged K5.39 to the uncharged alanine resulted in strong decreased
affinities of acrivastine and levocetirizine, but not meclozine and tripolidine which are their analogs
that lack the carboxy moiety (Wieland, Laak et al. 1999). Interestingly, the mutation of K5.39 to the
positively charged arginine resulted in a wild type phenotype again, demonstrating the importance
of the positive charge of K5.39 position for an interaction with the negatively charged carboxylate
group of acrivastine and levocetirizine (Wieland, Laak et al. 1999).
22 | P a g e

Introduction to the histamine H1 receptor
Figure 9. Binding of H1R antagonists to the H1R. The figure is obtained from Wieland (Wieland,
Laak et al. 1999).
(A) The H1R antagonist pharmacophore describing the position of the cis- and trans-aromatic rings.
(B) The H1R antagonist (cyproheptadine) was docked into the H1R model based on BR (ter Laak, Timmerman
et al. 1995). A view from the extracellular side shows the orientation of cyproheptadine between the TM
domain (yellow) and the ionic hydrogen bond interaction with Asp116 (D3.32) in TM III (TM3). The transaromatic
ring of cyproheptadine is in the proximity of Phe433 (F6.52), Phe436 (F6.55) of TM VI (TM6), and
the cis-aromatic ring is near Trp161 (W6.47) of TM IV (TM4).
Using a 3D model of the H1R receptor based on the crystal structure of bovine rhodopsin (ter Laak,
Timmerman et al. 1995) and site-directed mutagenesis analysis (Wieland, Laak et al. 1999),
Wieland and co-workers showed that residues W4.67 of TM4, F6.52 and F6.55 of TM6 are
responsible for the interaction with the aromatic rings of the H1R antagonists. The cis-ring of
cyproheptadine is located within 5 Å of a tryptophan W4.67 of TM4, whereas the trans-aromatic
ring of cyproheptadine is close to two phenylalanine residues F6.52 and F6.55 of TM 5 and a
tryptophan W1.67 of TM 4 (Figure 9) (Wieland, Laak et al. 1999). Removing the hydrophobic
phenylalanine at position 6.52 and 6.55 of the H1R showed a dramatic loss of affinity of
[ 3H]mepyramine, whereas replacing F6.55 and F6.52 with the hydrophobic but aliphatic
23 | P a g e

Chapter 1
methionine residue only slightly affected the binding of [ 3H]mepyramine, indicating that these two
phenylalanine residues in TM6 are most likely involved in a hydrophobic interaction with the H1R
antagonist (Wieland, Laak et al. 1999).
Active-state H1R
It is believed that several highly conserved residues are involved in the activation mechanisms of
the rhodopsin family of GPCRs, including the H1R. Bakker and co-workers (Bakker, Jongejan et al.
2008) have proposed that a conserved D2.50 of the NLxxxD motif in TM2 of the H1R forming a
hydrogen bond network with W6.48 of the CWxP motif in TM6, in the same manner as in rhodopsin
(Pardo, Deupi et al. 2007). This network constrains the toggle switch W6.48 in its inactive g+
conformation (Ruprecht, Mielke et al. 2004) and maintains the H1R in the inactive conformation
(Jongejan, Bruysters et al. 2005; Jongejan and Leurs 2005). N7.49 of the highly conserved NPxxY
motif in TM7 of the H1R is restrained toward TM6 in the inactive g+ conformation (Govaerts, Lefort
et al. 2001) by a water molecule (Bakker, Jongejan et al. 2008). The salt bridge interaction between
R3.50 of the highly conserved E/DRY motif in TM3 with its adjacent D or E3.49 residue and an
additional D/E 6.30 in TM6 (Alewijnse, Timmerman et al. 2000). Upon agonist binding, the
conformation of W6.48 was modified to point toward TM5. This occurs in the same concert manner
with the side chain at position S3.36 in TM3 of the H1R (Jongejan, Bruysters et al. 2005; Jongejan
and Leurs 2005). These conformation transitions of S3.36 and W6.48 disrupt the conserved
hydrogen bond network between W6.48 and D2.50 and consequently trigger N7.49 to adopt the
active (trans) conformation to interact with D2.50 (Jongejan, Bruysters et al. 2005; Jongejan and
Leurs 2005; Bakker, Jongejan et al. 2008). The conformational change of W6.48 also decrease the
proline kink of TM6, leading to the movement of the cytoplasmic end of TM6 away from TM3 and
disrupting the ionic interaction between R3.50 with D3.49 and E6.30 (Shi, Liapakis et al. 2002).
Bakker and co-workers also proposed that the physicochemical properties of the amino acid side
chain at position 6.40 in TM6 are important in the process of receptor activation (Bakker, Jongejan
et al. 2008). This position is located midway between the NPxxY motif and the ionic lock and it
seems to be a highly conserved hydrophobic residue in the rhodopsin family (L, 14%; V, 42%, I,
28% and M 5%). In the H1R, removal of the hydrophobic side chain (mutation I6.40A and I6.40G)
and introduction of positive charge (mutation I6.40R and I6.40K) or negative charge (mutation
I6.40E) resulted in the high constitutively active mutants (Bakker, Jongejan et al. 2008).
24 | P a g e

Aim of the thesis
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26 | P a g e
Aim of the thesis
Structure-based design is a powerful approach and a method of choice for lead optimization in drug
discovery, if X-ray structures of a target protein are available. Structure-based design is widely used
in drug discovery for soluble proteins, because they are straightforward to isolate, purify and
crystallize in comparisons to membrane proteins. Despite the importance of membrane proteins
such as GPCRs as drug targets, their structure determination has been very limited compared to
soluble proteins. The low success rate is due to the difficulties in obtaining large amounts of high
quality protein. Additionally, the purification of GPCRs also requires detergents, which often have a
negative effect on the protein stability and are also unfavorable during the crystallization process.
The main aim of the research described in this PhD thesis is to learn more about the structural
features of the H1R, a prototypical family A GPCR. In general, we employed approaches such as mass
spectrometry (MS) and site-directed mutagenesis analysis based on molecular modeling of the H1R.
The introduction to protein biochemistry, signal transduction pathways, and structural insight of
the family A of GPCRs and the H1R are discussed in Chapter 1. The expression and purification
strategies of GPCRs and the H1R is further discussed in Chapter 2. In Chapter 3, we show the use of
Baculovirus insect cell and the in vitro cell free expression systems for the production of the H1R
protein, followed by the purification and the MS analysis of the H1R. We employed the method
described for the protein production and MS analysis of the H1R subsequently for the human
histamine H4 receptor (H4R). Using MS technology, we further unraveled one of the glycosylation
sites of the H1R in Chapter 4, and investigated if this proteomic approach can be useful in the study
of ligand-receptor interaction. A preliminary result of covalent binding of an irreversible ligand
(phenoxybenzamine) to the H1R is presented in this chapter. Using mutagenesis together with
homology modeling studies, we showed the importance of the highly conserved TAS (I/V) motif in
TM3 and the residue I6.45 in the activation process of the H1R in Chapter 5 and Chapter 6,
respectively. Finally, Chapter 7 summarizes the conclusions of the work presented in this thesis
and the possible future experiments and developments are discussed.

CHAPTER
GPCRs expression and purification
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Chapter 2
28 | P a g e
Introduction to the expression and purification of GPCRs
Considering the crucial roles of G protein-coupled receptors (GPCRs) in many cellular and
physiological processes, the knowledge regarding their 3D structural details will facilitate the
understanding of how GPCRs function at atomic level (Pierce, Premont et al. 2002; Horn, Bettler et
al. 2003; Akermoun, Koglin et al. 2005; Lundstrom 2005). However, due to the low expression in
native tissues and the difficulties in purification and crystallization process of GPCRs (Sarramegna,
Talmont et al. 2003; Lundstrom, Wagner et al. 2006), the structural information of these important
membrane proteins has been limited for decades to crystal structures of the naturally abundant
GPCR, rhodopsin (Palczewski, Kumasaka et al. 2000; Edwards, Li et al. 2004). Although, this
rhodopsin crystals could provide significant advancements in GPCRs homology modeling, they are,
however, a light activated GPCR which somewhat differs from the other GPCRs that are activated by
ligands (Topiol and Sabio 2008). Recently, new crystal structures of GPCRs; the β1AR (Warne,
Serrano-Vega et al. 2008), the β2AR (Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al.
2007), the A2A adenosine receptor (Jaakola, Griffith et al. 2008) and the opsin (Park, Scheerer et al.
2008; Scheerer, Park et al. 2008)) have finally been obtained. These structures will provide the
opportunity for evaluating an actual ligand-mediated GPCR in structure based design. However,
much more high resolution information is needed, for example the structures of GPCRs from
different families, structures bound with different ligands, and structures of GPCRs in complex with
G proteins and other signaling molecules (Kobilka and Schertler 2008). In this chapter, we will
discuss many aspects regarding the production of GPCRs in various heterologous expression
systems, the techniques used in the purification and reconstitution processes towards
crystallography.
Recombinant expression of GPCRs
With the exception of rhodopsin, GPCRs express naturally at low level (concentration range of 20-
100 fmol/mg protein) (Lefkowitz 2007). Therefore, they need to be heterologously over-expressed
in the hosts such as Escherichia coli (E.coli), yeast, insect cells, or mammalian cells. The advantages
and disadvantages of each expression system are summarized in Table 1. It is generally difficult to
predict which expression system is best for the high level production of the targeted GPCRs. The
right combination between the target receptor and expression host must be found most likely by
trial and error (Grisshammer 2006; Wagner, Bader et al. 2006; McCusker, Bane et al. 2007).

GPCRs expression and purification
However, as shown in Table 2, it is now possible to generate sufficient quantities of several GPCRs
for crystallization or other biophysical studies purpose.
Table 1
Advantages and disadvantages of each expression systems
Expression
systems
Advantages Disadvantages
E.coli Rapid construction generation
No native G-proteins
Inexpensive
Problems folding membrane proteins
Ease of use
Lack of post-translational modifications
Simple culture requirements
Non-native lipid environment
Rapid doubling time
Inclusion bodies require proper re-
Variety of different strains (e.g. DH5α,
BL21, CAG627, KS474)
Produce inclusion bodies
Possibility in using labeled amino acids or
stable isotopes
folding
Yeast Rapid construction generation
Non-native glycosylation
Inexpensive
Simple culture requirements
Extensive genetics
Eukaryotic processing
Non-native lipid environment
Insect cells More native lipid environment than yeast Cost
Large scale culture applicable
Non-native glycosylation
Good safety profile
Non-native lipid environment
Time required to generate virus
Mammalian cells Native lipid environment
Cost
Native post-translational modifications Time required to establish stable cell
Stable cell lines provide consistent lines
expression level
Transient transfected cells required large
Transient transfected cells is fast
amount of DNA
Cell free Simple and Fast
Cost
High expression
Open system
Lack of post-translational modifications
Prokaryotic: Escherichia coli (E.coli)
E.coli is the most commonly used expression system. The major advantages of the E.coli expression
system are its low cost, ease of use, variety of different strains, its rapid doubling time and a straight
forward incorporation of specific labeled amino acids or stable isotopes. As a result of these
features, large scale protein production can be easily achieved (McCusker, Bane et al. 2007; Midgett
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Chapter 2
and Madden 2007). A number of high-resolution structures have been obtained for soluble proteins
recombinantly expressed in bacteria; however, it has been less successful in the case of membrane
proteins such as GPCRs. Because of their transmembrane topology, membrane proteins are highly
toxic to the bacterial host cells and therefore restricted their growth and reducing the protein yields
(Lundstrom 2007). Heterologous expression of eukaryotic membrane proteins can also be
hampered by a different synthesis, targeting, insertion and folding characteristics in the prokaryotic
host. For example, the polypeptide elongation and protein folding rates are considerably higher in
prokaryotes than in eukaryotes, which can cause mistargeting and misfolding of heterologously
expressed membrane proteins (Tate 2001). Also the lack of post-translation modifications in E.coli
may provide both pros and cons. Homogenous protein is more suitable for crystallization, but some
GPCRs (e.g. somatostatin, rhodopsin, β2AR, human thyrotropin and the bombesin BB2 receptors)
requires glycosylation for high affinity ligand binding and/or G-protein coupling (Rands, Candelore
et al. 1990; Rens-Domiano and Reisine 1991; Russo, Chazenbalk et al. 1991; Kaushal, Ridge et al.
1994; Kusui, Benya et al. 1994) and others (e.g. angiotensin II type 1 and follicle stimulating
hormone receptors) require glycosylation for transport to the plasma membrane (Davis, Liu et al.
1995; Deslauriers, Ponce et al. 1999; Jayadev, Smith et al. 1999; Dong, Filipeanu et al. 2007).
However, it has been shown also that eukaryotic membrane proteins that are glycosylated in
mammalian cells, such as the human CB2 receptor can still be functionally over-expressed in E.coli
(Yeliseev, Wong et al. 2005). Another disadvantage of the E.coli system is the difference in the
composition of the membranes between prokaryotes and eukaryotes. For example, the expression
of the mammalian serotonin transporter and the binding properties of oxytocin and human μ-
opioid receptors are dependent on cholesterol, which is not present in prokaryotic membranes
(Hasegawa, Loh et al. 1987; Gimpl, Klein et al. 1995; Tate 2001). However, the addition of certain
lipids during and/or after purification can sometimes restore functionality of over-expressed
membrane proteins (Bogdanov, Umeda et al. 1999). Nevertheless, E.coli has been successfully used
to express active GPCRs by implementing a combination of several strategies. The fusion of the
E.coli periplasmic maltose binding (PMB) protein to the N-terminus of GPCRs helps targeting the
expressed protein to the periplasmic membrane (which corresponds to the outer side of a
eukaryotic plasma membrane) as reported for the human adenosine A2A, rat neurotensin, human
serotonin 5HT1a, rat NK2, muscarinic M1 and M2, human β2AR and the human cannabinoid CB2
receptors (Bertin, Freissmuth et al. 1992; Grisshammer, Little et al. 1994; Hulme and Curtis 1998;
Furukawa and Haga 2000; Hampe, Voss et al. 2000; Weiss and Grisshammer 2002; White, Trinh et
al. 2004; Yeliseev, Wong et al. 2005). Also, the use of other fusion proteins such as a Bacillus subtilis
30 | P a g e

GPCRs expression and purification
integral membrane protein (Mistic) (Roosild, Greenwald et al. 2005), and thioredoxin (TrxA)
(Tucker and Grisshammer 1996) have been reported to be a useful fusion protein for GPCR
expression. Another approach has been to produce large amounts of eukaryotic membrane proteins
as inclusion bodies in E.coli. The protein in inclusion bodies is more resistant to proteolytic
degradation and easier to purify. However, to successfully obtain GPCRs with this approach, a
proper re-folding of protein is required. This technique has only been successful for the olfactory
OR5 receptor, the human leukotriene B4 receptor and the human serotonin 5HT4a receptor (Kiefer,
Krieger et al. 1996; Baneres, Martin et al. 2003; Baneres, Mesnier et al. 2005).
Other prokaryotic hosts
In addition to E.coli, there are several other bacterial expression systems for the production of
GPCRs. Unlike in E.coli system, the protein expressed in a gram-positive bacteria Lactococcus lactis
could be transported to the cytoplasmic membrane, therefore, circumventing the forming of
inclusion bodies (Monne, Chan et al. 2005). The L. lactis has been used successfully for the
expression of prokaryotic proteins such as the ABC transporter, and the yeast mitochondrial carrier
proteins CTP1 and AAC3 (Kunji, Slotboom et al. 2003). However, the expression of eukaryotic
membrane protein such as the human KDEL receptor in L. lactis only resulted in a yield less than
0.1% of the total membrane protein (de Ruyter, Kuipers et al. 1996). Bacillus subtilis, another Gram-
positive bacterium, has been considered as a good host for expressing large quantities of
recombinant interferon (Palva, Lehtovaara et al. 1983) and human cystatins (Akiba, Hayashi et al.
2006). However, its high endogenous protease activity has limited the use of this system
(Lundstrom 2007). Halinobacterium salinarum has become an alternative prokaryotic expression
host. Various membrane proteins such as the E.coli aspartate transcarbamylase, the yeast α mating
factor receptor, the human muscarinic M1, the serotonin 5-HT2 receptor and the adrenergic α2B
receptor have been expressed from H. salinarum (Turner, Reusch et al. 1999; Bartus, Jaakola et al.
2003). However, the GPCRs were expressed at low level. Interestingly, the expressed adrenergic α2B
receptor showed functional binding activity, although with 10 times lower binding affinities than in
mammalian cells (Bartus, Jaakola et al. 2003). Recently, due to its advantage in providing more
membrane surface per cell compared to other typical expression host, the photosynthetic
bacterium Rhodobacter sphaeroides has also been introduced to express some GPCRs such as the
human adenosine A2a, the human angiotensin AT1a and the human bradykinin B2 receptors (Roy,
Shukla et al. 2008).
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Chapter 2
Yeast
Yeasts strains are valuable candidates for heterologous protein expression. Like E.coli, they are well
characterized, easy to culture at low cost and straightforward to manipulate genetically. An
important feature of yeast cells is also that they possess a G protein which activates a MAP kinase
cascade, similar to that found in mammalian cells. Therefore, heterologous receptors expressed in
yeast cells can be coupled to this endogenous signaling pathway, potentially leading to a functional
response (Francken, Luyten et al. 1997; Midgett and Madden 2007). Furthermore, yeasts are
eukaryotic cells and have the potential to perform most types of post-translational modifications
and form disulfide bonds, similar to more complex eukaryotic cells. Yet, glycosylation is mostly of
the high-mannose type and appears limited for heterologous membrane proteins (Eckart and
Bussineau 1996; Midgett and Madden 2007). However, yeast with humanized glycosylation
machineries has also been introduced and may provide an important alternative expression system
(Hamilton, Davidson et al. 2006). It is also worthy to note that yeast cells are surrounded by a cell
wall that may hinder the recovery of non-secreted proteins such as GPCRs (Francken, Luyten et al.
1997; Midgett and Madden 2007). Moreover, the low cholesterol content of the yeast membranes
may result in altered ligand binding properties of some GPCRs, although, co-solubilization with
mammalian lipids such as cholesterol has been shown to stabilize the active conformation of the μ-
opioid receptor (Opekarova and Tanner 2003). The yeast systems that are widely used for
heterologous protein expressions are Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces
pombe (S. pombe) and Pichia pastoris (P. pastoris). The S. cerevisiae or baker’s yeast has been used
extensively to express GPCRs, for example, the human A2A receptor fused with C-terminal green
fluorescent has been highly expressed (~4 mg functional protein per litre of culture) in S. cerevisiae
(Niebauer and Robinson 2006). The S. pombe or fission yeast is less commonly used. Yet, it has
recently been examined as it showed the ability to express higher complexity eukaryotic proteins
(Giga-Hama and Kumagai 1998). Comparison of GPCR expression demonstrated that the human
dopamine D2 receptor was expressed at 5-fold higher levels in the S. pombe than in the S. cerevisiae
(Sander, Grunewald et al. 1994). The methylotrophic yeast P. pastoris is capable of achieving higher
cell densities than the yeast S. cerevisiae, therefore, allowing production of g/L quantities of
recombinant proteins. 100 GPCRs were expressed in P. pastoris, for which a large number of GPCRs
could reach a high expression level (1-10 mg/L). The highest level (180 pmol/mg receptor protein)
was measured for the human adenosine A2a receptor (Lundstrom, Wagner et al. 2006). It has also
been shown that the expression of the human opioid receptor in P. pastoris could be improved by
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GPCRs expression and purification
fusing the S. cerevisiae yeast α-factor signal sequence to their N-terminal tails (Sarramegna, Muller
et al. 2005).
Insect cells
The use of baculovirus vectors for the infection of insect cell lines is the second most commonly
used expression system aster E.coli. The baculovirus expression system (BVES) emerged from
studies designed to control insect pests (Smith, Summers et al. 1983). However, the system has
gained more widespread use as a method for protein expression. The BVES requires the
introduction of the foreign GPCR gene of interest into a non-essential region of the viral genome,
usually Autographa california nuclear polyhedrosis virus (AcNPV), by homologous recombination
with a transfer plasmid (McCusker, Bane et al. 2007). The production of protein is produced when
insect cells are infected with the recombinant virus (Fraser 1992). Most of the cells that are
commonly used are derived from ovarian tissue of the fall army worm Spodoptera frugiperda (Sf9
and Sf21). Although the E.coli and yeast systems are less expensive to culture, the advent of serum-
free media for growing of Sf9 or Sf21 cell lines and the use of intermediate-scale shaker suspension
have made the insect cells more widely accessible (Midgett and Madden 2007). Insect cells provide
a correct folding of recombinant protein as well as disulfide bond formation and perform post
translational modifications (such as fatty acid acylation and phosphorylation) identical to those in
mammalian cells, except for glycosylation. The oligosaccharides produced in insect cells are smaller
and of the high-mannose type. However, a characterization of a new insect cell line, Trichoplusia ni
(Tni, High Five), showed that this cell line is able to produce sugars of the complex type (Ogonah,
Freedman et al. 1995). Sf9 cells could also express certain Gα subunits indicating their ability to
provide favorable interactions for GPCRs (Kleymann, Boege et al. 1993; Mulheron, Casanas et al.
1994; Obosi, Schuette et al. 1996; Kuhn and Gudermann 1999). Consequently, GPCRs that are
expressed in insect cells system are almost always able to bind to their respective ligands
(McCusker, Bane et al. 2007). A drawback to the baculovirus system is the requirement for high
titer viral stocks to infect cell cultures. Productions of these stocks are time-consuming and
laborious to perform (McCusker, Bane et al. 2007). Another issue is the quality of protein crystal
because insect cells often give heterogeneous populations of receptors due to variable post-
translational modifications (Reilander, Boege et al. 1991). Moreover, insect plasma membranes are
not identical to those of mammalian cells. They have higher amounts of unsaturated lipids and
lower amount of cholesterol (Marheineke, Grunewald et al. 1998), which can alter ligand binding
(Gimpl, Klein et al. 1995). However, once the tedious start up process has finished, the insect cell
33 | P a g e

Chapter 2
system is easy to scale up and has produced numerous GPCRs (as shown in Table 1) (Grisshammer
and Tate 1995; Massotte 2003; Ratnala, Swarts et al. 2004; Akermoun, Koglin et al. 2005; Cherezov,
Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007; Sansuk, Balog et al. 2008; Warne, Serrano-
Vega et al. 2008). After several attempts, the crystals of ligand-mediated GPCR have finally been
obtained by using insect cells as an expression system. The β2AR crystals were made by forming a
complex with an antibody Fab fragment (Rasmussen, Choi et al. 2007) or by fusing in the middle of
the third ICL3 to T4 lysozyme (Cherezov, Rosenbaum et al. 2007). The turkey β1AR was mutated to
enhance thermo stability over the wild type receptor and finally yielded a good crystal of 2.7Å
(Warne, Serrano-Vega et al. 2008) and the A2A adenosine receptor protein was stabilized through
the T4 lysozyme insertion (Jaakola, Griffith et al. 2008).
Mammalian cells
Among heterologous expression systems, mammalian expression systems, although costly and
time-consuming, clearly offers the most native cellular environment for expression of GPCRs.
Mammalian cells contain necessary co-receptors, providing the optimal membrane composition
and can perform the right post translational modifications (McCusker, Bane et al. 2007).
Heterologous protein expression in mammalian cells may be achieved through transient or stable
expression and the use of viral vectors. Recently, Standfuss and co-workers have reported a crystal
structure of a rhodopsin mutant produced from transfected COS-7 cells. The final yield of purified
protein is about 2.5 mg of rhodopsin from 50 plates (15 cm) of COS-7 cells (Standfuss, Xie et al.
2007). Some mammalian cell lines such as HEK293 and CHO cells are able to be stably transfected
and to grow in suspension. However, the expression levels obtained are often low and it is difficult
to obtain reliable stable cell lines due to cell toxicity in the presence of high levels of GPCRs; for
example the β2-adrenergic and μ-opioid receptors could not be maintained in suspension culture
(Stanasila, Pattus et al. 1998; Kohno, Fukushima et al. 2000). To overcome these limitations,
inducible mammalian cell systems have been developed (Tate 2001; Neddermann, Gargioli et al.
2003; Lipscomb, Mowry et al. 2004; Weber, Rimann et al. 2004; Ivanova, Brandli et al. 2005; Bach,
Grigat et al. 2007). Recently, it was found that the tetracycline-inducible system enabled a codon-
optimized hamster β2-adrenergic receptor to be expressed at levels of 220 ± 40 pmol/mg protein
(Chelikani, Reeves et al. 2006). Another approach to produce high levels of GPCRs is the use of viral
vectors, such as the Semliki Forest Virus (SFV), to infect mammalian host cells. In many cases,
relatively high yields of protein have been obtained, such as adenosine A2A receptors with the
expression level of 287 pmol/mg protein (Hassaine, Wagner et al. 2006). Besides SFV, it has also
34 | P a g e

GPCRs expression and purification
been shown that baculoviruses are able to infect mammalian cells (Hofmann, Sandig et al. 1995;
Shoji, Aizaki et al. 1997). This approach has recently been used for large scale protein production in
mammalian cell expression system (Scott, Modha et al. 2007) by employing the pfastbacmam-1
vector to generate a recombinant baculovirus in which the polyhedrin promoter is replaced with a
cytomegalovirus (CMV) promoter (Condreay, Witherspoon et al. 1999). Both the generation of
stable cells containing GPCRs of interest and the handling of viral stocks can be laborious and time
consuming. As a result, with the development of serum-free media and of relatively cheap
transfection reagents, large-scale transient Transfection (LSTT) of mammalian cells has been used
as an alternative expression method (Derouazi, Girard et al. 2004; Geisse, Jordan et al. 2005; Baldi,
Hacker et al. 2007).
Cell Free Expression System
Cell Free Expression is an alternative method that allows full control and high flexibility of
conditions to produce protein of interest without intact cells. The protein synthesis can be
performed either in a batch reaction using a test tube or another container with a fixed volume or
in a continuous exchange reaction. In the batch mode, the translation of protein stops as soon as the
substrate is finished, or any product or by-product reaches an inhibitory concentration, usually
within 20 to 60 minutes of incubation. Due to these limitations (e.g. a short lifetime and low
productivities for a preparative scale), a continuous cell free expression systems was first
introduced in 1988 by Spirin and co-workers (Spirin, Baranov et al. 1988). The principle of this
system was the continuous addition of reaction substrates (amino acids and nucleoside
triphosphates) with continuous removal of reaction products through a porous membrane during
incubation. This design could produces hundreds of micrograms per ml reaction volume. Recently
several modified versions of the Spirin system have been reported (Kigawa and Yokoyama 1991;
Kawarasaki, Kawai et al. 1995; Kim, Kigawa et al. 1996; Kigawa, Yabuki et al. 1999). Kigawa and co-
workers showed that by using a dialysis membrane to facilitate the continuous supply of substrate
and removal of by-products, an E.coli coupled transcription-translation system yields as much as 6
mg protein per ml reaction volume (Kigawa, Yabuki et al. 1999). Besides the feasibility to produce
milligram of proteins, Cell Free Expression system also offers a solution for traditional in vivo
expression problems such as cell-induced protein degradation and aggregation, loss of DNA
template, and toxicity (Kigawa, Yabuki et al. 1999). However, this system can be costly and it lacks
GPCR-interacting signaling components and post translational modifications, resulting in the
misfolding of proteins (McCusker, Bane et al. 2007). The E.coli cell free expression system was
35 | P a g e

Chapter 2
successfully used for stable-isotope labeling of proteins for NMR spectroscopy such as amino acid-
selective 15N labeling (Kigawa, Muto et al. 1995), dual-amino acid selective 13C- 15N labeling (Yabuki,
Kigawa et al. 1998), and uniform 13C/ 15N-labeling (Kigawa, Yabuki et al. 1999). Another cell free
expression system has also been developed from the wheat embryos. They possess numerous
advantages such as low cost, easy availability in large amounts, and low endogenous incorporation
(Madin, Sawasaki et al. 2000). This system has been applied for high throughput functional
proteomics (Sawasaki, Ogasawara et al. 2002). More than 50 genes can be translated in parallel,
yielding between 0.1-2.3 mg of protein in 2 days. The wheat germ cell free expression system also
offers the capacity to synthesize high molecular weight proteins and is therefore more suitable for
the expression of eukaryotic proteins. However, up to now expression of GPCRs using cell free
systems is still limited and depends on fusion partners. By adding an N-terminal thioredoxin tag,
the human β2 adrenergic, rat muscarinic acetylcholine, rat neurotensin and human histamine H1R
have been expressed using E.coli cell free expression systems (Ishihara, Goto et al. 2005; Sansuk,
Balog et al. 2008). A N-terminal T7 tag was essential for expression of the porcine vasopressin
receptor (Klammt, Schwarz et al. 2005). In all cases, GPCRs were produced as either precipitates or
detergent soluble protein by the addition of detergents to the reaction mixture. Expression levels
have been reported as high as milligrams per milliliter of reaction mixtures. However, the
radioligand binding results of GPCRs produced from cell free expression system have been reported
for only β2AR (Ishihara, Goto et al. 2005) and histamine H1R (Sansuk, Balog et al. 2008).
Transgenic animals for protein production
The first crystal structure of GPCR was successfully obtained by simply isolating rhodopsin from
bovine retina(Palczewski, Kumasaka et al. 2000). This success took advantage of the naturally
enriched levels of rhodopsin that occur in retina and the fact that this protein is uniformly
glycosylated to provide large amounts of homogenous protein for crystallization. To further apply
these advantages for the expression of other GPCRs, some laboratories have been exploited the
retina’s rod cells from several animal species as a “bioreactor” to produce nascent heterologous
GPCRs for purification and crystallization. Proof of this concept has been reported with the fruit fly
(Drosophila melanogaster) (Eroglu, Cronet et al. 2002) , the African clawed toad ( Xenopus laevis)
(Zhang, Salom et al. 2005), and the mouse (Li, Salom et al. 2007; Salom, Wu et al. 2008). The fruit fly
D. melanogaster has a high biogenesis capacity for membrane proteins and sufficient space in the
membrane to accommodate the over-expressed protein (Wagner, Bader et al. 2006). They have
been successfully used to over-express the D. melanogaster metabotropic glutamate receptor
36 | P a g e

GPCRs expression and purification
(Eroglu, Cronet et al. 2002). The use of X. laevis for protein expression is still limited to the small
scale since it requires injection of in vitro synthesized mRNA into each individual oocyte (Zhang,
Salom et al. 2005). The attempts to replace endogenous retinal rhodopsin in mouse eyes with a
heterologous GPCR such as adenosine A1 receptor and 5HT4 were reported (Li, Salom et al. 2007;
Salom, Wu et al. 2008). However, the amount of receptor protein presents in the engineered retina
was still less than what they have expected. The additional efforts in improving homozygote lines
and extraction-purification protocol are required (Li, Salom et al. 2007; Salom, Wu et al. 2008).
Purification of GPCRs
In general, the purification of membrane protein such as GPCRs is not as easy as the purification of
water-soluble proteins, although the methods used are generally the same. The first purification
step is the preparation of a receptor-enriched fraction, followed by the solubilization of the GPCRs
from the membrane compartment. Once suitable conditions for solubilization have been identified,
a robust and effective purification scheme is required. Since purification of membrane proteins
must be achieved in the presence of detergents in order to maintain the solubility of the
hydrophobic proteins, the application of standard chromatographic procedures is not so straight
forward (Sarramegn, Muller et al. 2006). Charged steroid-based detergents, such as sodium cholate
or deoxycholate, will prevent the binding of proteins to ion-exchange or dye matrix columns. Non-
ionic detergents such as dodecyl maltoside which are more suitable in many cases are not
compatible with the use of ammonium sulphate. However, once the protein is bound to a column, it
is possible to exchange detergent simply by washing the column with a buffer containing a different
detergent (Gwyn 1994). In addition to this, the number of column steps should be kept to a
minimum to preserve receptor integrity at maximum yield (Grisshammer, White et al. 2005).
Finally, in order to study membrane proteins in a stable environment, the solubilized and purified
proteins need to be reconstituted back into an artificial environment that mimics their native
membranes.
The solubilization step
The objective of the solubilization step is to extract membrane proteins from their lipid
environment using detergents. Detergents are amphiphillic molecules that can disrupt the lipid
bilayer by dispersing the lipids and membrane protein into detergent micelles (Sarramegna, Muller
et al. 2005). As shown in Table 3, there are various kinds of detergents. Non-ionic detergents
37 | P a g e

Chapter 2
contain uncharged hydrophilic head groups of either polyoxyethylene or glycosidic groups (e.g.
Triton X-100, DM, digitonin).
Table 2
Summary of the GPCRs protein production from various expression systems
Expression
System
38 | P a g e
Year References GPCRs Species Expression
level
Purified
Protein
E. coli 1988 Marullo, 1998 β2 Human 0.2% ND
1992 Bertin, 1992 5-HT1A human ND ND
1993 Haendler, 1993 ETB human ND ND
1993 Grisshammer, 1993 NTS1 rat 15 pmol/mg ND
1994 Ho, 2008 NK2 rat ND ND
1994 Jockers, 1994 A1A human 0.4 pmol/mg ND
1996 Tucker, 1996 NTS1 rat 15 pmol/mg ND
1996 Kiefer, 1996 OR5 ND 10% 20 mg/L
1997 Grisshammer, 1997 NTS1 rat 13 pmol/mg ND
2000 Furukawa, 2000 M2 human 0.2nmol/0.1L ND
2000 Hampe, 2000 β2 human 6 pmol/mg ND
2001 Busuttil, 2001 TSH human ND 2.5 mg/L
2002 Weiss, 2002 A2A human 20 nmol/L ND
2003 Baneres, 2003 BLT1 human 10 mg/L 2-3 mg/L
2004 White, 2004 NTS1 rat 0.45 mg/L 0.2 mg/L
2005 Yeliseev, 2005 CB2 human 1-2 mg/L 1 mg/L
2005 Baneres, 2005 5HT4 mouse 5-8 mg/L 0.5 mg/L
2006 Krepkiy, 2006 CB2 human 15 pmol/mg ND
2007 Harding, 2007 NTS1 rat ND ND
S. cerevisiae
S. pombe
2007 Bane, 2007 NK1 human ND 5 mg/L
1996 Mollaaghababa, 1996 Rho bovine 2 mg/L 40 μg/L
1998 Andersen, 1998 D1 human 13 pmol/mg ND
2001 Liitti, 2001
α 2C human 25 pmol/mg ND
2007 O’Malley, 2007 α2A Human 4 mg/L 1 mg/L
1995 Ficca, 1995 Β2 human 7.5 pmol/mg ND
1995 Arkinstall, 1995 NK2 human 1.2 pmol/mg ND
1996 Talmont, 1996 Μ human 0.4 pmol/mg ND
P. pastoris 2001 Schiller, 2001 ETB human 45 pmol/mg 15pmol/mg
2002 Feng, 2002 CB2 human ND ND
2004 De Jong, 2004 D2 human 13 pmol/mg ND
2005 Kim, 2005 CB1 human 3.6 pmol/mg ND
2005 Sarramegna, 2005 Μ human 30 mg/L 12 mg/L
2006 Andre, 2006 DRD2 human 39.1 ND
2006 Andre, 2006 NK1 human 24 ND

GPCRs expression and purification
Table 2 (continued)
Expression Year References GPCRs Species Expression
System
level
2006 Andre, 2006
NK2 human 120
2006 Andre, 2006 5-HT5A human 39.1
2006 Andre, 2006
human 76
Insect cells
HEK293
Α2A
Purified
Protein
ND
ND
ND
2007 Li, 2007 CB2 human ND ND
2008 MAgnin, 2009 H1 human ND 0.5 mg/L
1991 Parker, 1991 M1 human 2 nmol/l 0.3 nmol/L
1991 Parker, 1991 β2 turkey 2 nmol/l 0.15 nmol.L
1991 Parker, 1991 M2 Human 10-15 nmol/l 4.5-7 nmol/l
1991 Reilander, 1991 β2 human 12-19 ND
1993 Kwatra, 1993 β2 human 7 ND
1993 Kwatra, 1993 NK1 rat 1-2 ND
1995 Kwatra, 1995 β2 human 5-20 1-4 nmol/l
1995 Kobilka, 1995 β2 human 2.5 5 nmol/l
1996 Hayashi, 1996 M2 human 10.8 nmol/l 4.4 nmol/l
1996 Nekrasova, 1996 OR4 human ND ND
1998 Ohtaki, 1998 PAC1 human 50-150 250 µg/l
1999 Klaassen, 1999 Rho bovine 4 mg/l 0.5-1 mg/l
2000 Ghanouni, 2000 β2 human ND ND
2000 Hampe, 2000 β2 human 17 ND
2003 Warne, 2003 β1 turkey 7.5 mg/l 2.5 mg/L
2003 Park, 2003 M2 human ND ND
2003 Panneels, 2003 Glu Dros 11 ND
2003 Bodor, 2003 P2Y12 human ND 0.3-0.5 mg/L
2004 Ratnala, 2004 H1R human 4-7 mg/l 3-4 mg/L
2004 Asmann, 2004 Secretin human 830 1.4 mg/L
2005 Akermoun, 2005 H1 human 85 pmol/mg ND
2007 Rasmussen, 2007 β2 human ND 8-12 mg/L
2007 Cherezov, 2007 β2 human ND 5 mg/L
2008 Serrano-Vega, 2008 β1 turkey ND 2.5 mg/L
2008 Jaakola, 2008 A2A human ND 3 mg/L
2008 Ruan, 2008 TP human ND 4 mg/0.3 L
2002 Reeves, 2002 Rho bovine 10 mg/l 9 mg/l
2002 Vicentic, 2002 α1A, B, D human 0.15-3.6 ND
2003 Christoffers, 2003 μ mouse 10 ND
2005 Christoffers, 2005 Δ mouse 10 ND
2006 Gan, 2006 PTH1 human ND ND
2006 Chelikani, 2006 β2 human 50 μg/plate 12 μg/plate
2007 Daulat, 2007 MT1-2 human ND ND
2007 Standfuss, 2007 Rho human ND 3mg/50plate
COS-7 2003 Sen, 2003 Α2B human 176 pmol/mg ND
2007 Hassaine, 2006 Α2A human 287 pmol/mg ND
39 | P a g e

Chapter 2
Table 2 (continued)
Expression Year References GPCRs Species Expression Purified
System
level Protein
Cell free 2005 Ishihara, 2005 β2 human 1 mg/ml ND
2007 Klammt, 2007 ETB human 3 mg/ml ND
2007 Klammt, 2007 V2R human 3 mg/ml ND
2008 Sansuk, 2008 H1 human 3-5 mg/ml 0.2 mg
Drosophilla 2002 Eroglu, 2002 DmGluRA Dros 170µg/g fly ND
melanogaster
heads
Xenopus 2005 Zhang, 2005 5-HT1A human 1-5
ND
laevis
ng/tadpole
Mouse 2007 Li, 2007 A1 Human 90 ng/eye ND
2008 Salom, 2008 5-HT4 human 100 ng /eye ND
Abbreviations: α,β, adrenergic receptor; 5-HT, hydroxytryptamine receptor; ET, endothelin receptor; NTS,
neurotensin receptor; NK, neurokinin receptor; OR, olfactive receptor; TSH, thyroid-stimulating hormone
receptor; BLT, leukotriene receptor; CB, cannabinoid receptor; A, adenosine receptor; TP, tromboxane
receptor; M, muscarinic receptor; D, dopamine receptor; Rho, rhodopsin; μ-, δ-opioid, opioid receptor; PAC,
pituitary adenyl cyclase-activating polypeptide receptor; Glu, metabolic glutamate receptor; P2Y, purinergic
receptor; MT, melatonin receptor; V, vasopressin receptor; PTHR, parathyroid hormone receptor; H,
histamine receptor; DmGluRA, Drosophila metabotropic glutamate receptor; Dros, Drosophila; ND, not
mentioned
They are mild and non-denaturing as they break lipid-lipid and lipid-protein interactions rather
than protein-protein interactions (Seddon, Curnow et al. 2004). This allows membrane proteins to
be solubilized without affecting the structural features of the protein (Sarramegna, Muller et al.
2005). Ionic detergents contain either a head group with a net charge that can be cationic (e.g.
cetyl-trimethyl-ammonium bromide) or anionic (e.g. SDS), together with a hydrophobic chain or a
polar and an apolar face (e.g. sodium cholate). Among ionic detergents, SDS is effective in
solubilizing membrane proteins but is also known to denature the protein (Sarramegna, Muller et
al. 2005). However, some membrane proteins can be re-natured from SDS by transferring the
protein to another detergent or lipid environment (Paulsen, Finkenzeller et al. 1993; Booth, Flitsch
et al. 1995; Lau and Bowie 1997). Prior to a purification step, a suitable condition for solubilization
is required for achieving maximum solubilization efficiency and stability of functional receptors in
solution (Tucker and Grisshammer 1996; Warne, Chirnside et al. 2003). The detergent of choice
must be made on a trial-and-error basis for each individual protein. The main criteria are the
compatibility of the chosen detergent with the purification steps and the necessity to preserve the
functionality of the receptor during the solubilization and purification steps (Sarramegna, Muller et
al. 2005). The efficiency of detergents can be quantified by determining the receptor protein
content in the solubilized sample before and after a centrifugation step at 100,000 x g. Radioactive
40 | P a g e

GPCRs expression and purification
ligands have been used to bind to the receptor and enable the quantification (Mazina, Strader et al.
1996; de Jong, Grunewald et al. 2004). However, this technique detects only the functional fraction
of the solubilized receptors, since in the presence of detergents, numerous proteins tend to lose
their ability to bind ligands (Sarramegna, Muller et al. 2005). Another important aspect in choosing
detergents is the ease of removal of the detergents during a reconstitution step. In this case,
detergents with a high critical micellar concentration (CMC) will be easier to eliminate
(Sarramegna, Muller et al. 2005). However, detergents with low CMC can also be removed
efficiently over a short period of time using polystyrene beads (Levy, Bluzat et al. 1990; Sansuk,
Balog et al. 2008). Detergents with a low CMC such as digitonin, CHAPS and DM are used generally
for GPCRs solubilization, since they give a good efficiency of solubilization and a better stability of
protein (le Maire, Champeil et al. 2000). Some GPCRs require the use of several detergents; for
example, neurotensin receptors were solubilized with an efficiency of 85% when using a mix of
detergents of CHAPS, DM and Cholesterol hemisuccinate (Tucker and Grisshammer 1996).
The Purification step
There are several different chromatographic methods which can be used to purify membrane
proteins based on their properties. The most common protein purification technique for GPCRs is
the affinity chromatography called Immobilized metal affinity chromatography (IMAC)
(Janssen, Bovee-Geurts et al. 1995; Ratnala, Swarts et al. 2004; Sarramegna, Muller et al. 2005;
Sansuk, Balog et al. 2008). The IMAC purification relies on the specific binding of the targeted
protein to an immobilized phase. In order to do this, the targeted protein should be produced as a
fusion protein of N- and/or C-term to an affinity tag. Among numerous numbers of affinity tags, the
histidine tag is the most commonly used tag (Arnau, Lauritzen et al. 2006). The histidine tag is very
short and therefore, does not alter the solubility properties and rarely affect the characteristic of
the targeted protein (Graslund, Nordlund et al. 2008). During the purification step, the imidazole
rings in the histidine tag bind to the divalent metal ions attached to an immobile phase, and enable
the separation of the targeted protein from the other proteins or impurities by applying several
washing steps. The targeted protein can be eluted from the immobilized phase by using a gradient
of imidazole or by decreasing the pH of the buffer. In general, IMAC purification is performed either
in phosphate buffer (pH 8) or HEPES buffer (pH 7.5-8.0). An ionic strength, which maintains the
solubility and stability of the proteins, should be equivalent to 300-500 mM NaCl. Adding glycerol
(~10%) in the buffer also aids the solubility and stability of the proteins. Reducing reagents such as
41 | P a g e

Chapter 2
dithiothreitol (DTT) should also be included in the buffer to maintain a reducing environment and
finally, a small amount of imidazole in the buffer helps reducing the binding of less histidine-rich
proteins to the IMAC column (Graslund, Nordlund et al. 2008).
Table 3
General properties of a range of common detergents
Detergent type CMC (1) Comments
Non-ionic detergents
Triton X-100
n-octyl-glucoside
n-dodecyl-β-D-maltoside
(DM)
Ionic detergents
Sodium dodecyl sulfate (SDS)
Sodium cholate
42 | P a g e
0.24
14.5
0.16
3.5
13.0
A mild detergent that is usually non-denaturing. Give a strong
absorbance at 280 nM. Difficult to remove by dialysis owing to
very low CMC. However, can be removed using BioBeads.
Expensive to use for initial solubilization but good for
reconstitution step owing to its high CMC.
A mild detergent that can stabilize protein that is sensitive in
solubilized form. It has a longer alkyl chain length than n-octyl
glucoside, resulting in a lower CMC making this difficult to be
removed.
Denaturing to most membrane proteins. Commonly used in PAGE
analysis.
A non-denaturing detergent, but can disrupt protein-protein
interactions in oligomeric complexes. Not compatible with ionexchange
column chromatography.
Zwitterionic detergents
CHAPS
4.0 Electrically neutral detergent with similar structure to Sodium
cholate, but compatible with ion-exchange column
chromatography.
(1) CMC; Critical Micelle Concentration; the concentration of detergents/surfactants at which micelles
spontaneously start forming.
To date, there is a broad array of resins used in the IMAC system such as TALON Metal Affinity
(Clontech), the QIAexpressionist (QIAGEN) and HisTrap HP (GE Healthcare). Besides batch
purification, the purification step can also be integrated by high-performance liquid
chromatography (HPLC) system, such as ÄKTA from GE Healthcare. The IMAC purification has
several advantages such as the strong and specific binding between the protein and the
immobilized phase, the mild elution conditions and the ability to control selectivity (by including
low concentrations of imidazole in the chromatographic buffers). Furthermore, the purity of the
protein obtained from the IMAC can be easily optimized by controlling the ratio of recombinant
protein to the column size (Graslund, Nordlund et al. 2008). Another affinity chromatography
technique is the Lectin affinity chromatography, which separates the protein based on the

GPCRs expression and purification
affinity of proteoglycans for lectin. Only glycosylated protein is bound to the lectin resins and eluted
with monosaccharides, α-D-methyl mannoside, α-D-methyl glycoside, or N-acetyl glucosamine
(Cummings and Kornfeld 1982). There are different kinds of lectins available for the purification of
glycoproteins. Concanavalin A (ConA) or lentil lectin binds with a very high affinity to proteins
presenting N-glycosylated amino acids with high-mannosidic carbohydrates. Wheat germ
agglutinin (WGA) lectin selectively interacts with proteins containing complex N-glycans. The
human β2AR expressed in Sf9 cells bind to ConA resin, but does not interact with a WGA resin,
showing that the receptor mainly contains high-mannosidic sugars (Reilander, Boege et al. 1991).
The rat M3 receptor expressed in Sf9 cells was able to bind to both WGA and ConA, demonstrating
heterogeneity in glycosylation (Cummings and Kornfeld 1982), which underlines a limitation of this
chromatographic method. In Biotin-Streptavidin affinity chromatography, the protein is fused
with a 15-24 amino acid sequence called bio-tag. The tagged protein is biotinylated by biotin-
protein ligase and subsequently purified using the affinity of biotin for avidin or Streptavidin
(Sarramegn, Muller et al. 2006). A successful example is the purification of the NTS1 neurotensin
receptor by Tucker and Grisshammer. The receptor protein is C-terminally fused with the Bio-tag
and the protein is purified on an avidin column. The final yield is 38% with a remarkable 60-fold
purification (Tucker and Grisshammer 1996). Besides these affinity chromatography techniques,
Ionic Exchange Chromatography is also a useful technique for protein purification. In this
method, the protein can be separated on cation or anion exchange columns depending on their
isoelectric point and the pH of the buffer (Sarramegn, Muller et al. 2006). Most of the protein
purifications are done on anion exchange columns because proteins are generally negatively
charged at physiological pH values (pH 6-8). The proteins are absorbed to the column, made from
beads of inactive material such as cellulose or dextran, and then eluted from the column by
adjusting the salt concentration or changing of the pH. The proteins are eluted according to the
tenacity with which they bind. Those that are the most highly charged at that pH will bind the
tightest. An example of this method is the purification of several GPCRs such as β2AR and
muscarinic receptors by Parker and co-workers (Parker, Kameyama et al. 1991). Ion-exchange
chromatography can also be performed as an additional purification step, if the target protein is
contaminated with other proteins. For example, different phosphorylated states of protein can
sometimes be resolved using ion-exchange chromatography (Bullock, Debreczeni et al. 2005).
Since proteins can occur in many different sizes; we can therefore use this property for the
purification purpose. The separation based on the protein size is called Gel filtration
43 | P a g e

Chapter 2
chromatography or Size exclusion chromatography. This type of purification is more generic
and can be performed in any buffer conditions. This technique has been used to resolve the
oligomerization state of the target protein (Graslund, Nordlund et al. 2008) or it is often used as a
complement to other methods (Parker, Kameyama et al. 1991; Mazina, Strader et al. 1996; Tucker
and Grisshammer 1996; Christoffers, Li et al. 2003; Christoffers, Li et al. 2005). Finally, after the
protein purification step, samples should be subjected to SDS-PAGE analysis. The purity and the
size of protein can be investigated after staining the SDS gel with a dye such as Coomassie Brilliant
blue (Bradford 1976). To further quantify the amount and concentration, The purified proteins can
be measured at UV A280 (Graslund, Nordlund et al. 2008) or by a Bradford protein determination
assay (Bradford 1976) to further quantify the amount and concentration. Proper storage of the
purified protein is another important aspect. The purified protein should be frozen in liquid
nitrogen and stored at -80°C. It may be necessary to store the proteins in small aliquots to avoid
damaging freeze-thaw cycles. If the proteins are found to be inactivated after one freeze-thaw cycle,
they can be stored at 4°C for short periods of time or at -20°C in high concentration of glycerol, or
as an ammonium sulfate suspension (Graslund, Nordlund et al. 2008).
The reconstitution step
An ideal situation for the reconstitution of membrane protein such as GPCRs is to have an
environment that more closely resembles the natural lipid bilayer. One of the strategies is the
incorporation of the GPCRs into liposomes. Liposomes can be made from a variety of lipids, such as
phosphatidylserine, phosphatidylcholine, cholesterol, E.coli lipids or asolectin (Katzen, Peterson et
al. 2009). Reconstitution of protein into such liposomes can be achieved by the addition of the
appropriate lipid-detergent micelles into the detergent-solubilized GPCRs, followed by the removal
of detergent from the homogenous mixed solution (Gwyn 1994; Remigy, Caujolle-Bert et al. 2003).
The detergent is dialyzed out over several hours, resulting in the formation of phospholipid vesicles
(Gwyn 1994). However, this method requires a long dialysis process and cannot be used with low
CMC detergents such as sodium cholate, and CHAPS (Chattopadhyay and London 1984). The
dilution of the ternary mixture is a simpler variation of the dialysis method and has been shown to
produce reconstituted membrane protein efficiently (Almog, Kushnir et al. 1986; Dolder, Engel et al.
1996; Remigy, Caujolle-Bert et al. 2003). Yet, this method is again only possible when using high
CMC detergents and it does not allow complete detergent removal (Rigaud, Mosser et al. 1997).
Finally, a hydrophobic adsorption of the detergent to polystyrene beads (BioBeads) can be used to
remove low CMC detergents (Akiba, Toyoshima et al. 1996; Rigaud, Mosser et al. 1997; Sansuk,
44 | P a g e

GPCRs expression and purification
Balog et al. 2008). The drawbacks of this method are its (too) fast detergent removal and the
adsorption of protein or lipid onto beads (Remigy, Caujolle-Bert et al. 2003). However, the
reconstitution process may not always be straightforward. If the detergent binds more strongly to
the protein than to the lipids, the protein will be in its soluble form. Therefore, a further detergent
removal will precipitate the protein unless it is able to integrate into preformed lipid vesicles. In
addition to this, the protein will also aggregate if it releases the detergent more readily than the
lipid does. Thus, an optimized reconstitution process should be carefully developed to have the
mixture consists mainly of ternary detergent-protein-lipid micelles (Remigy, Caujolle-Bert et al.
2003). The success of the reconstitution process can be monitored by dynamic light scattering
(Engel, Hoenger et al. 1992; Dolder, Engel et al. 1996), turbidimetry (Almog, Kushnir et al. 1986;
Ollivon, Eidelman et al. 1988), and cryo-electron microscopy (Lambert, Levy et al. 1998). The most
common method for detergent removal is dialysis (Jap, Walian et al. 1991; Kuhlbrandt, Wang et al.
1994). For an optimized reconstitution process, the lipid-protein ratio (LPR) should be in the range
of 0.2–2, and the protein concentration should not be less than 1 mg/ml (Remigy, Caujolle-Bert et
al. 2003). Moreover, the choice of the detergent is critical. It must preserve the native state of the
protein during solubilization and purification, and possess the correctly balanced binding affinities
to the protein and the lipid. In addition to this, the measurement of the detergent concentration
prior to the reconstitution process is necessary since excess of detergent may destabilize
membrane protein complexes, leading to a loss of the ternary structure (Remigy, Caujolle-Bert et al.
2003). Furthermore, the selection of lipids is also of importance. In general, proteins exhibit
optimal activity in artificial membranes with a lipid composition that most closely reflects that of
their native membranes (Gwyn 1994). For example the recovery of a D. melanogastor metabotropic
receptor was dependent on the presence of ergosterol, the sterol originally found in Drosophila
membrane (Gimpl and Fahrenholz 2002). It has also been shown that the kinetic of β2AR
conformational changes can be improved by careful selection of lipids used for artificial membrane
(Yao, Parnot et al. 2006). All of these studies emphasize the importance of the reconstitution step to
provide the most closed-to-native environment for purified GPCRs. Recently a new platform of
membrane environment has been developed. This membrane platform is so called the recombinant
High Density Lipoprotein (rHDL) membrane or “nanodisc”. The nanodiscs are nanometer-sized,
discoidal particles comprising amphipathic helical proteins that wrap themselves around the planar
circumference of a lipid bilayer (Borch and Hamann 2009; Katzen, Peterson et al. 2009). In contrary
to the liposomes, nanodiscs are mono-disperse, highly stable and could provide a better
representation of the phase transition behavior of membrane proteins such as GPCRs (Borch and
45 | P a g e

Chapter 2
Hamann 2009; Cappuccio, Hinz et al. 2009). Up to now, there are several membrane proteins
including the β2AR (Leitz, Bayburt et al. 2006) that have been successfully incorporated into such
membrane discs (for further review see ref (Borch and Hamann 2009)).
46 | P a g e
High resolution structures of GPCRs
One of the most challenging tasks in membrane protein structural studies is to obtain highly
diffracting crystals of GPCRs. Since, most of the GPCRs are expressed, except rhodopsin, at low
levels in native tissues (Alkhalfioui 2009; Rosenbaum, Rasmussen et al. 2009; Topiol and Sabio
2009). Therefore, a suitable recombinant expression system must be developed to generate
natively folded membrane proteins. As shown in Table 4, only Sf9 insect cells (Rasmussen, Choi et
al. 2007; Hanson, Cherezov et al. 2008; Jaakola, Griffith et al. 2008; Scheerer, Park et al. 2008;
Warne, Serrano-Vega et al. 2008) and COS-7 mammalian cells (Standfuss, Xie et al. 2007) have
produced enough purified GPCRs for structure determination. For bovine rhodopsin, a high level of
expression in native rod-cell disc membranes directly allows purification from a natural source
(Palczewski, Kumasaka et al. 2000; Murakami and Kouyama 2008; Park, Scheerer et al. 2008;
Scheerer, Park et al. 2008; Shimamura, Hiraki et al. 2008). Another challenge in GPCRs
crystallography is to overcome the protein stability problems. GPCRs have generally poor thermal
stability and their extra-membranous loops regions are prone to proteolysis (Topiol and Sabio
2009). Various methods have been used to enhance the thermal stability of the GPCRs, including the
use of ligands to stabilize the receptor (Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al.
2007; Hanson, Cherezov et al. 2008; Jaakola, Griffith et al. 2008; Warne, Serrano-Vega et al. 2008);
the use of stabilizing mutants (Cherezov, Rosenbaum et al. 2007; Hanson, Cherezov et al. 2008;
Warne, Serrano-Vega et al. 2008); the addition of lipids during purification and crystallization
(Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007; Jaakola, Griffith et al. 2008), and
having a high salt concentration (Jaakola, Griffith et al. 2008). Furthermore, various methods have
also been used to enhance the proteolytic stability, including the truncation of disordered regions
(Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007; Jaakola, Griffith et al. 2008; Warne,
Serrano-Vega et al. 2008), the fusion of a stable protein at the ICL3 (Cherezov, Rosenbaum et al.
2007; Jaakola, Griffith et al. 2008), and the complex formation with an antibody Fab fragment
(Rasmussen, Choi et al. 2007). The crystallization of dark-state rhodopsin structures did not require
such modifications due to their high stability (Palczewski, Kumasaka et al. 2000; Salom, Lodowski
et al. 2006; Standfuss, Xie et al. 2007; Murakami and Kouyama 2008; Shimamura, Hiraki et al.
2008). However, to obtain the active state opsin crystal (Park, Scheerer et al. 2008; Scheerer, Park

GPCRs expression and purification
et al. 2008), the protein needed to be crystallized at low pH, since this condition is known to
stabilize the active conformation of opsin (Topiol and Sabio 2009). After obtaining large quantities
of purified and stable GPCRs, an effort in coaxing GPCRs into crystals is further needed. For
detergent-solubilized membrane protein such as GPCRs, the absence of exposed polar surface area
outside the micelle can be a major limitation to crystallization (Alkhalfioui 2009; Rosenbaum,
Rasmussen et al. 2009). Using of the antibody Fab complex approach [26] or the fusion of T4
lysozyme strategy (Cherezov, Rosenbaum et al. 2007; Jaakola, Griffith et al. 2008) provided has
provided sufficient polar surface in obtaining the crystals of adrenergic and adenosine receptors.
All these approaches give a promising sign that it is possible to obtain other similar engineered
GPCR structures. However, when using these alternative strategies, it is important to characterize
the engineered GPCRs for their native-like pharmacology and biophysical properties (Rosenbaum,
Rasmussen et al. 2009). The high resolution X-ray structures of GPCRs will enhance our
understanding of the mode of action of agonists, inverse agonist and antagonists, and thereby
accelerated drug discovery efforts aimed toward this class of membrane protein.
Recombinant expression of the H1R protein
Like the majority of GPCRs, the H1R proteins are found in small quantities in native membranes
such as cat small intestinal muscle (Takagi and Uchida 1970), calf thymocytes (Osband and
McCaffrey 1979), guinea pig brain (Toll and Snyder 1982), and rat liver (Fukui, Wang et al. 1988).
In order to obtain a sufficient amount of the H1R protein for structural studies such as
crystallography or NMR, it is necessary to express the recombinant protein in expression hosts. Up
to date there are reports showing the high expression level of H1R proteins in insect cells (Ratnala,
Swarts et al. 2004; Akermoun, Koglin et al. 2005), yeast P. pastoris (Magnin, Fiez-Vandal et al. 2009)
and the in vitro cell free expression system (Kamonchanok, Balog et al. 2008). Ratnala and co-
workers reported in 2004 the functional expression of the H1R in insect Sf9 cells with at least 1000-
fold higher than in native tissues (Ratnala, Swarts et al. 2004). The H1R protein was further
extracted by dodecyl-maltoside purified using affinity chromatography (IMAC) and reconstituted
into artificial membrane proteins made from asolectin. In the end, they were able to obtain up to 4-
7 mg of the purified H1R protein from 1 Liter culture (Ratnala, Swarts et al. 2004). The milligram
quantities of purified H1R have been subsequently used in their structural studies with NMR
(Ratnala, Kiihne et al. 2007).
47 | P a g e

Chapter 2
Table 4
Crystal structures of GPCRs
GPCRs Species PDB Å Modifications In complex with Expression
system
bovine 1F88 (1) Rhodopsin
2.8 None 11-cis-retinal Native
Rhodopsin
48 | P a g e
bovine 2I35 (2)
2I36 (2)
2I37 (2)
3.8
4.1
4.1
Deprotonated Rhodopsin 11-cis-retinal Native
bovine 2J4Y (3) 3.4 Mutation at N2C/D282C and
N15D
squid 2ZIY (4)
2Z73 (5)
3.7
2.5
Deletion of 75 residues at
the C-term
11-cis-retinal COS-7
cells
11-cis-retinal Native
bovine 3CAP (6) 2.9 None None Native
bovine 3DQB (7) 3.2 Contain synthetic Gα at the
C-term
None Native
β2AR human 2RH1 (8) 2.4 Deletion of 48 residues at
the C-term
β2AR human 2R4R (9)
2R4S (9)
3.4
3.4
-Deletion of 48 residues at
the C-term
-Mutation at N187E
-Fusion with T4-lysozyme in
place of ICL3
β2AR human 3D4S (10) 2.8 -Deletion of 65 residues at
the C-term
-Fusion with T4-lysozyme in
place of ICL3
-Mutation at E122W
β1AR Turkey 2VT4 (11) 2.7 -Deletion of 30 residues at
the N-term
-Deletion of 28 residues in
IC3
-Deletion of 100 residues at
the C-term
-Various mutations to
increase thermal stability
A2A
Receptor
Human 3EML (12) 2.6 -Deletion of 96 residues at
the C-term
-Fusion with T4-lysozyme in
place of ICL3
- Carazolol
- ICL3 is bound
with Antibody
Fab fragment
Insect
cells
Carazolol Insect
cells
Timolol
Cholesterol
Insect
cells
Cyanopindolol Insect
cells
ZM241385 Insect
cells
References: (1) Palczewski, Kumasaka et al. 2000, (2) Salom, Lodowski et al. 2006, (3) Standfuss, Xie et al. 2007,
(4) Shimamura, Hiraki et al. 2008, (5) Murakami and Kouyama 2008, (6) Park, Scheerer et al. 2008, (7) Scheerer,
Park et al. 2008, (8) Cherezov, Rosenbaum et al. 2007, (9) Rasmussen, Choi et al. 2007, (10) Hanson, Cherezov
et al. 2008, (11) Warne, Serrano-Vega et al. 2008, (12) Jaakola, Griffith et al. 2008

GPCRs expression and purification
In 2005, Akermoun and co-workers (Akermoun, Koglin et al. 2005) reported the characterization of
16 GPCRs, including the H1R, in various type of insect cells such as Sf9, Sf21 and High five. The
highest expression level of the H1R is observed in Sf21 insect cells after 72h of infection.The
expressed H1R protein has the Bmax of 85 pmol/mg protein when binding to [ 3H]mepyramine
(Akermoun, Koglin et al. 2005). Magnin and co-workers have developed a rapid and generic
purification process of various GPCRs expressed in P. pastoris. This purification relies on the
presence of a C-terminal biotinylation domain in the recombinant proteins, which binds with a high
affinity to streptavidin-agarose beads. After the washing step, the specific tobacco etch virus (TEV)
protease was added to remove the C-terminal tag and the native protein is released from the
agarose beads. Using this method, they were able to obtain around 0.5 mg of the purified H1R from
a liter culture of P. pastoris (Magnin, Fiez-Vandal et al. 2009). Recently, we also reported the use of
an in vitro cell free expression system to express the H1R protein (Kamonchanok, Balog et al. 2008).
The cell free technology has several advantages as compared with the technology based on living
organisms. These are the speed and directness of all procedures, the absence of constraints from
living cells and the wide possibilities of product modifications. Using the cell free technology we
were able to rapidly produce the sufficient amount of the H1R protein for the mass spectrometric
analysis. These results will be further discussed in the Chapter 3.
49 | P a g e

CHAPTER
Mass spectrometric analysis of the H1R:
glycosylation and binding of an
irreversible H1R antagonist
Kamonchanok Sansuk 1, Crina Balog 2, Anne van der Does 2, Raymond
Booth 3 , Wim de Grip 4 , Andre Deelder 2 , Remko Bakker 1 , Paul
Hensbergen 2 , Rob Leurs 1
1 Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit Amsterdam,
Department of Medicinal Chemistry, De Boelelaan 1083, 1081HV, Amsterdam, the Netherlands
2 Biomolecular Mass Spectrometry Unit, Leiden University Medical Center (LUMC), Department
of Parasitology, P.O. Box 9600, 2300 RC, Leiden, the Netherlands
3 Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville,
Florida 32610-0485
4 Department of Biophysical Organic Chemistry, LIC/LACDR, Gorlaeus Laboratories, Leiden
University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands
5 Current address: Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach, Germany
Jib
[Type the company name]
(Part of this research is published in: J Proteome Res, 2008, 7(2), 621-9)
[Pick the date]

Chapter 4
72 | P a g e
Introduction
G protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome. They
are membrane proteins with seven transmembrane (TM) domains that mediate responses to a
broad range of stimuli such as light, taste, odor, hormones and neurotransmitters. GPCRs have been
implicated in numerous human disorders and diseases, thus making them attractive targets for
drug discovery. It is estimated that nearly half of all modern drugs regulate GPCR activity in some
way (Jacoby, Bouhelal et al. 2006; Schlyer and Horuk 2006; Lefkowitz 2007; Warne, Serrano-Vega
et al. 2008). Despite their success as drug targets, the structural insight at the atomic level has long
been limited to the crystal of rhodopsin (in its inactive state) (Palczewski, Kumasaka et al. 2000).
Recently, three new GPCR proteins (Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007;
Jaakola 2008; Warne, Serrano-Vega et al. 2008) and the activated state of bovine rhodopsin have
been successfully crystallized (Park, Scheerer et al. 2008). These crystals provide significant new
insight in GPCRs structures and their activation. However, similar information is still needed for
other GPCRs that have not been successfully studied by X-ray crystallography yet. To date, there are
a variety of alternative techniques to detect receptor-ligand interactions. Mass spectrometry (MS)
analysis has gained wide acceptance for the characterization of proteins. Proteins can be rapidly
analyzed in the intact form or after the digestion with selected enzymes. Detection of the intact
protein provides molecular mass information and the extent of protein heterogeneity. The analysis
of proteolytic peptides allows confirmation of the amino acid sequence and a thorough
investigation of any post-translational modification. With its high sensitivity, together with
continuous improvements in technology and computer software, MS analysis has become not only a
key player in protein sequencing but also a tool for the characterization of higher order protein
structures, non-covalent complexes and receptor-ligand binding interactions (Sinz 2007). Up till
now, there are several GPCRs, such as rhodopsin (Ball, Oatis et al. 1998; Ablonczy, Kono et al. 2001;
Kraft, Mills et al. 2001; Ablonczy, Crouch et al. 2005), the tachykinin NK-1 receptor (Alves, Sachon
et al. 2007), the cannabinoid CB2 receptor (Zvonok, Yaddanapudi et al. 2007), the neurotensin NTS1
receptor (Ho, White et al. 2008) and the histamine H1R (Sansuk, Balog et al. 2008) that had their
protein sequences determined (almost) completely by MS analysis. We are particularly interested
in the histamine H1R, one of the prototypic members of the family A GPCRs, which has been used as
a model system for our GPCR production, purification and MS analysis (Sansuk, Balog et al. 2008).
Using the developed protocol as described in Chapter 3, we have identified for the first time an
asparagine residue in the H1R protein that is subjected to N-linked glycosylation. Furthermore, we

MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
also showed preliminary data of the study of ligand-H1R binding interactions using
phenoxybenzamine, an irreversible H1R antagonist.
Reagents and Materials
Experimental Procedures
Standard laboratory chemicals, including phenoxybenzamine, were purchased from Sigma
Chemical Co. (St. Louis, MO). Insect-Xpress insect medium, penicillin, and streptomycin were
purchased from Bio Whittaker. Ni-NTA Agarose resin, NuPAGE Novex Bis-Tris Gel, and SimplyBlue
Safestain were purchased from Invitrogen.
Site-directed mutagenesis of the H1R
The single point mutant human histamine H1R (N5Q H1R mutant) was created using a PCR-based
mutagenesis approach (Cormack 2001). The constructs were sub-cloned into the expression vector
pcDEF3 and verified by DNA sequencing.
Cell culture and transfection
COS-7 African green monkey kidney cells were maintained at 37 °C in a humidified 5% CO2/95% air
atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 50 IU/ml penicillin, 50
µg/ml streptomycin and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected
using the DEAE-dextran method as previously described (Bakker, Schoonus et al. 2001).
Reporter-gene assay
Reporter-gene assays were performed essentially as described previously (Bakker, Schoonus et al.
2001). In brief, cells transiently co-transfected with pNF-κB-Luc (125 µg/1.10 7 cells) and pcDEF3
encoding WT or N5Q H1R mutant (25 µg/1.10 7 cells) were seeded in 96 well white plates (Costar)
in serum free culture medium and incubated with drugs. After 48 hrs, cells were assayed for
luminescence by aspiration of the medium and the addition of 25 µl/well luciferase assay reagent
(0.83 mM ATP. 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 µM Na2H2P2O7, 38.9 mM Tris (pH 7.8).
0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100 and 2.6 µM dithiothreitol). After 30 min,
luminescence was measured for 3 sec/well in a Victor plate reader (Wallac).
73 | P a g e

Chapter 4
Baculovirus Expression System and the treatment of recombinantly expressed H1R protein with
phenoxybenzamine
Spodoptera frugiperda ovarian cells (Sf9) were cultured as monolayer at 27 ºC in tissues culture
flasks in a serum-free insect medium supplemented with 50 units/mL penicillin and 50 mg/mL
streptomycin. Under this conditions, the cell doubling time was typically 20-24 h. Cells were
infected with pBac-H1His10 virus at a multiplicity of infection (MOI) of 0.1. Infected Sf9 cells were
maintained in a serum-free insect medium supplemented with 5 μM Leupeptin, 50 units/mL
penicillin and 50 mg/mL streptomycin and harvested at 5 days post infection (dpi). For the pre-
incubation with phenoxybenzamine, the infected Sf9 cells were incubated with phenoxybenzamine
at the concentration of 30-90 nM in Bis-Tris propane buffer (20 mM Bis-Tris propane, 1 M NaCl, 1
mM Histidine, 5 mM β-mercaptoethanol, and 5 µM Leupeptin, pH 7.8). After 30 min incubation, the
Bis-Tris propane buffer containing phenoxybenzamine was removed and the infected Sf9 cells were
washed with fresh Bis-Tris propane buffer for at least 3 times.
Radioligand Binding Assays
The infected Sf9 cells or the transfected COS-7 cells were harvested and homogenized in the H1R
binding buffer (50 mM Na2/K Phosphate buffer, pH 7.4). The membranes were then incubated with
5 nM [ 3H] mepyramine (specific activity 30 Ci/mmol) (Perkin-Elmer) in a final volume of 200 µL for
30 min at 25 °C. Nonspecific binding was determined by incubation with 1 µM of the selective H1R
antagonist mianserin. Non-bound radioactivity was removed by filtration through Whatman GF/C
filters that had been treated with 0.3% polyethyleneimine. Filters were washed 3–4 times with ice-
cold H1R binding buffer, and radioactivity retained on the filters was measured by liquid
scintillation counting. Binding data was evaluated by a nonlinear, least-square, curve-fitting
procedure using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).
Protein purification of the H1R protein
The infected Sf9 cell membranes was prepared based on the protocol according to Ratnala and co-
workers (Ratnala, Swarts et al. 2004). The membranes were subsequently solubilized in Bis-Tris
propane buffer, containing 2% (w/v) n-dodecyl-β- D-maltoside (DDM), under gentle rotation at 4 °C
for 4 h. The supernatant was separated by ultracentrifugation at 80,000 g for 20 min at 4 °C and
incubated with 2 mL Ni-NTA agarose resin, pre-equilibrated with Bis-Tris propane buffer. After
overnight incubation at 4 °C, Ni-NTA agarose resin was settled by gravity, and the unbound lysate
74 | P a g e

MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
was aspirated. The resin was washed with Bis-Tris propane buffer, containing 0.2% (w/v) DDM,
and imidazole (concentration 20 and 100 mM). The bound H1R proteins were eluted from the Ni-
NTA agarose resin with Bis-Tris propane buffer, containing 0.2% (w/v) DDM and 200 mM
imidazole. Purified H1R protein was analyzed by gel electrophoresis, followed by protein staining or
Western blotting.
Gel Electrophoresis and Western Blotting
Protein samples were mixed with SDS-PAGE sample buffer (2% (w/v) sodium dodecyl sulfate, 0.04
M dithiothreitol, and 0.015% bromophenol blue in 0.5 M Tris-HCl, final pH 6.8 to a final volume of
25–50 µL. Samples were run on a NuPAGE Novex Bis-Tris Gel at 200 V constant, according to the
manufacturer’s protocol. For immunodetection, proteins were blotted onto a nitrocellulose
membrane (1 h at 100 V) in ice-cold blot buffer (25 mM Tris and 0.2 M glycine in 20% methanol).
Blots were subsequently immunoassayed for the presence of His-tagged receptor, using anti-His
antibody (1:2000 dilution) (Qiagen). Protein staining was performed using Coomassie G-250 stain
(SimplyBlue Safestain).
In-Gel Tryptic Digestion and Deglycosylation
Protein bands were excised from a NuPAGE Novex Bis-Tris gel, cut into small pieces, and washed
with 25 mM NH 4HCO 3 followed by dehydration with 100% acetonitrile (ACN) for 10 min. For
reduction and alkylation, dried gel particles were first incubated with 10 mM dithiothreitol for 30
min at 56 °C. Following dehydration with ACN, gel plugs were subsequently incubated in 55 mM
iodoacetamide for 20 min at room temperature. After washing with 25 mM NH 4HCO 3 and
dehydration with 100% ACN, the gel particles were completely dried in a centrifugal vacuum
concentrator (Eppendorf, Hamburg, Germany). For in-gel digestion using RapiGest TM SF, dried gel
particles were re-swollen in a 1% RapiGest TM SF solution (in 25 mM NH 4HCO 3) followed by 10 min
incubation at 37 °C. Excess solution was removed, and the gel particles were dried in a centrifugal
vacuum concentrator. Dried gel particles were subsequently re-swollen on ice and incubated with
10-15 μL of a trypsin solution (12.5 ng trypsin/μL 25 mM NH4HCO3) for 10 min at 37 °C. Following
tryptic digestion, the overlaying digestion-solution was collected. After that, 0.5% trifluoroacetic
acid (TFA) (final concentration) was added to the digestion solution followed by incubation for 40
min at 37 °C. The sample was centrifuged at 20,000 g, for 10 min. Finally, the resulting supernatant
was collected, and the pellet was extracted two times with 7.5 µL of 70% isopropanol. For
subsequent deglycosylation, the supernatant was adjusted to pH 8 using ammonium bicarbonate
75 | P a g e

Chapter 4
and incubated with 5 Units of PNGase F (Roche Diagnostics, Mannheim, Germany) overnight at 37
°C.
MALDI-ToF MS
MALDI-ToF(-ToF) analyses were performed on an Ultraflex II mass spectrometer (Bruker Daltonics,
Bremen, Germany) using dihydroxybenzoic acid (5 mg/ml in 50%ACN/0.1% TFA) as a matrix. The
mass spectrometer was used in the reflector ion mode. For fragment ion analysis in the tandem
time-of-flight (ToF/ToF) mode, precursors were selected in a timed ion gate. Fragment ions
generated by laser-induced decomposition of the precursor were further accelerated in the LIFT
cell, and their masses were analyzed after the ion reflector passage.
Nano-LC-Iontrap MS
The samples containing the digested H1R in 70% isopropanol were concentrated to approximately
1–2 µL in a centrifugal vacuum concentrator. Subsequently, 7 µL of 70% ACN/0.1%TFA was added,
and the sample was sonified for 10 min. Next, 0.1% TFA was added until a concentration of 20%
ACN/0.1% TFA was reached. This was then shortly sonified (2 min) just prior to injection onto the
nanoLC system (Ultimate, Dionex-LC Packings, The Netherlands). The sample was separated on a
C18 pepmap100 column (75 µm i.d., 15 cm long; Dionex-LC packings, Amsterdam, The Netherlands)
coupled with an electrospray ionization (ESI)-iontrap mass spectrometer (HCTultra, Bruker
Daltonics, Bremen). The samples were injected onto the same system as described previously in the
chapter 3 of this thesis (see experimental procedures section) the samples were eluted using a
linear gradient of 0% solvent B (95% ACN/0.1% formic acid) up to 60% solvent B in 60 min.
MS/MS analysis was performed in a data-dependent manner using the five most intense peaks in an
MS spectrum.
Analysis of H1R deglycosylation
76 | P a g e
Results and Discussion
The analysis of GPCR glycosylation have relied mainly on indirect methods such as apparent
molecular weight shifts after Western blotting of tunicamycin-treated cells or PNGase- treated
protein extracts (Kim, Zhang et al. 2005; Zhang, Kim et al. 2007). Moreover, site-directed
mutagenesis of asparagines within N-glycosylation consensus sites has yielded valuable
information, not only about the exact localization of the glycan moiety, but also on the functional

MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
consequences (Rands, Candelore et al. 1990; Kohno, Wada et al. 2002). Because of the common
difficulties in proteomic analysis of GPCRs, studies applying MS techniques for the direct
assessment of the glycosylation sites in GPCRs are restricted to rhodopsin (Duffin, Lange et al.
1993; Barnidge, Dratz et al. 1999; Abdulaev and Ridge 2000; Nakagawa, Miyamoto et al. 2001;
Ablonczy, Goletz et al. 2002). As described in Chapter 3, the H1R protein obtained from the
baculovirus/insect cell expression system appeared as a smear (see Chapter 3, Figure 1) possibly
reflecting H1R glycosylation. Within the amino terminal domain, the H1R contains 2 sequences
(N5SS and N18KT) that fit with the consensus sequence for N-glycosylation (Asn-Xaa-Ser/Thr, Xaa
being any amino acid except proline). It is hitherto unknown whether these sites are actually
glycosylated. To address this question, we first analyzed a tryptic digest of H1R before and after
treatment with PNGase F, using MALDI-ToF MS. PNGase F cleaves asparagine N-linked
oligosaccharides at the β-aspartyl-glycosylamine linkage, converting asparagine into aspartic acid.
Within the PNGase F-treated sample, we identified a peptide at m/ z 1405.7 which was absent in
the untreated sample (Figure 1A). MALDI-ToF-ToF analysis revealed that it corresponds to the
acetylated N-terminal tryptic peptide of the H1R containing an aspartic acid at position 5. This
aspartic acid corresponds to the converted asparagine (N) within the consensus sequence Asn-Ser-
Ser after PNGase F releases, identifying this N5 as a glycan attachment site. However, in these
experiments we did not observe glycosylation at N18. Although this probably reflects site-specific
glycosylation at N5, we cannot formally exclude the possibility of protein glycosylation at N18 as
well. N5 was not fully glycosylated since we also identified the non-glycosylated counterpart of this
peptide. To analyze the N5 glycosylation in more detail, we specifically searched for glycopeptides
within our LC-iontrap MS data through the analysis of the glycopeptide marker ion at m/ z 366
(hexose1HexNAc1 (Wuhrer, Catalina et al. 2007)) in our MS/MS spectra. Two major groups of
glycopeptides corresponding to different glycan structures attached to the Asn 5-containing tryptic
peptide were observed. The first contained a heterogeneous population of high mannose structures
(exemplified in Figure 1B). The other major group consists of N-glycans with terminal GlcNAc-
residues with or without fucosyl residues linked to the N, N′-diacetyl-chitobiose core (exemplified in
Figure 1C). These glycan structures were predicted based on what is known from glycosylation in
insect cells (Altmann, Staudacher et al. 1999). Since glycosylation in insect cells does not resemble
mammalian type of glycosylation, future MS analysis of H1R protein purified from insect Sf9 cells,
stably expressing mammalian glycosyltransferases (Mimic TM Sf9 insect cells), or purified from
mammalian cells overexpressing the H1R should be performed to reveal the mammalian
glycosylation pattern of the H1R protein.
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Chapter 4
Figure 1. Identification of the H1R glycosylation
Tryptic peptides from Sf9 cell produced H1R were deglycosylated using PNGase F and analysed by MALDI-
ToF MS. (A) A peptide at m/z 1405.7 was selected for MALDI-ToF-ToF and identified as the N-terminal
tryptic peptide from H1R containing as Asp residue at position 4 which corresponds to the Asn5 residue in
H1R. (B, C) Glycopeptides containing Asn5 were further analysed by LC-iontrap MS/MS. Red triangle, fucose;
blue square, N-acetylglucosamine; green circle, mannose.
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MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
Many GPCRs are glycosylated in vivo, and glycosylation has been shown to be implicated in proper
GPCR expression, signaling, and trafficking (Walsh, Foley et al. 1998; Wheatley and Hawtin 1999;
Zhang, Austin et al. 2001; Zvonok, Yaddanapudi et al. 2007). To further evaluate whether the
glycosylation at N5 plays a role in the expression, signaling and trafficking of the H1R, we therefore
mutated the asparagine into glutamine (N5Q H1R mutant). The N5Q H1R mutant was expressed in
COS-7 cells and its binding property was further evaluated with the radioligand [ 3H]mepyramine.
As shown in Figure 2A, the N5Q H1R mutant was well expressed in COS-7 cells with the Bmax of
12.4±9.7 pmol/mg protein (N=3) and has an affinity towards the radioligand [ 3H]mepyramine with
the KD of 0.9±0.1 nM (N=3), similar to that of the wild type H1R (Bmax = 14.8±4.6 pmol/mg protein,
N=7 and KD =1.5±0.2, N=7). This result demonstrates that the N-glycosylation at Asparagine 5 is not
essential for the H1R expression and the targeting of H1R protein to the plasma membrane. The N5Q
H1R mutant was also tested for the the functionality using NFκB reportergene assay. Figure 2B
shows that both the wild type and the mutant H1R still possess basal activity and could be
stimulated upon induction with its ligand histamine (pEC50 of the wild type H1R: 6.5±0.1, N=5;
pEC50 of the N5Q H1R mutant: 6.5±0.1, N=5 ). Furthermore, the inverse agonist mepyramine
inhibited the basal activity of both the wild type and the N5Q H1R mutant (pIC50 of the wild type
H1R: 7.9±0.1, N=3; pEC50 of the N5Q H1R mutant: 8.0±0.1, N=3).
Figure 2. Characterization of the N5Q H1R mutant, compared to the wild type H1R
(A) The wild type (�, dotted line) and the N5Q H1R mutant ( , straight line) were expressed in COS-7 cells
and futher analyzed for their expression level and KD values by radioligand binding analysis using
[ 3H]mepyramine. (B) The H1R constructs were expressed in COS-7 cells. Upon induction with histamine,
both the wild type (�, dotted line) and the N5Q H1R mutant ( , straight line) show the increasing NFκB
activation and the inverse agonist mepyramine was able to inhibit the basal response of both the wild type
(�, dotted line) and the N5Q H1R mutant (�, straight line).
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These results suggest that the mutation of the H1R at the N-linked glycosylation site (N5) did not
affect its functional coupling to Gq pathway. However, as mentioned earlier that the glycosylation of
the H1R is also occurred at the residue N18, it might be worthwide to characterize the H1R
constructs with a single mutation at N18 and double mutations at N5 and N18 for future studies on
the importance of N-linked glycosylation in the H1R.
MS analysis of phenoxybenzamine treated H1R
Phenoxybenzamine is known as a classical α-adrenergic receptor (α-AR) antagonist. However, the
clinical use of phenoxybenzamine has been limited due to its long lasting α-AR blockade. Besides
being known as a blocking agent for α-AR, phenoxybenzamine is also capable of inhibiting H1R
responses (Cook 1971; Cook 1988; Rappen-Cremer 1989; Takayanagi 1996).
Figure 3. Chemical structure of phenoxybenzamine
The structure of phenoxybenzamine and its reactive aziridinium intermediate in aqueous solution are
shown. The figure is obtained from Frang, et al. 2001 (Frang, Cockcroft et al. 2001).
Cook and co-workers have determined the effects of phenoxybenzamine on histamine-induced
contractions of isolated guinea pig ileum. They found that at a phenoxybenzamine concentration of
5.9 x 10 -7 M and an exposure time of 5 min, a shift of about a half log unit is observed without the
depression of the maximum response. When increasing the concentration, the maximum response
is depressed, and at the concentration of 5 x 10 -5 M and higher, the response is totally abolished
(Cook 1971). A similar inhibition profile of phenoxybenzamine is also observed in the work of
Takayanaki and co-workers. A progressive (> 70 min) treatment of guinea pig ileum tissue with
phenoxybenzamine at a concentration of 10 -6 M inhibits the response of H1R to histamine
(Takayanagi 1996). Furthermore, it has also been shown that phenoxybenzamine could block the
response induced by a partial agonist 2-(2-pyridyl)-ethylamine (PEA) (Cook 1988; Rappen-Cremer
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MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
1989). It is thought that in an aqueous solution, phenoxybenzamine forms an unstable aziridinium
ion (Figure 3) which can alkylate side chains of amino acid residues, such as –SH, -OH, =NH, and –
COOH, and resulting in an irreversible binding between the ligand and the target protein (Jenkinson
1996). It has also been reported that of the susceptible amino acid residues, cysteine with the –SH
side chain is the most reactive (Shulman-Roskes, Noe et al. 1998). Prior to the mapping of
phenoxybenzamine binding site on the H1R protein, we first tested whether phenoxybenzamine
would bind irreversibly to the H1R. In this case, we treated the recombinantly expressed H1R from
baculovirus/insect cells expression system with various concentration of phenoxybenzamine (30-
90 nM) for 30 min. The Bmax level of the recombinantly expressed H1R, compares to that of non-
phenoxybenzamine treatment, was measured afterwards by the radioligand binding analysis using
[ 3H]mepyramine. As shown in Figure 4A, the alkylating effect of phenoxybenzamine on the H1R
protein blocked the binding of [ 3H]mepyramine to the H1R by 53%, 77% and 98%, when treating
the receptor at the phenoxybenzamine concentration of 30, 60 and 90 nM, respectively.
Figure 4. Effect of the phenoxybenzamine treatment on the H1R protein
(A) The H1R protein expressed on Sf9 cells were incubated in the absence and presence (30, 60 and 90 nM)
of phenoxybenzamine (PB) for 30 min at 37 °C. (B, top) The H1R protein samples from purification process
were analyzed by western blot using anti-His antibody. The protein bands were present at 55 kDa. (B,
bottom) Bmax values of the H1R proteins were determined using Radioligand binding analysis; (1) H1R
membrane, (2) Phenoxybenzamine treated H1R membrane, (3) Phenoxybenzamine treated H1R after
solubilization, (4) Phenoxybenzamine treated H1R after purification.
A similar alkylating effect of phenoxybenzamine was also observed on the α-AR (Frang, Cockcroft et
al. 2001). Besides showing the irreversible binding effect of phenoxybenzamine on the H1R, these
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Chapter 4
data also suggest that phenoxybenzamine may have at least some part of its binding site in common
with H1R antagonists such as mepyramine (Cook 1988). In order to map the amino acid residue that
may be involved in the interaction of phenoxybenzamine and the H1R protein using MS analysis, we
further purified the phenoxybenzamine-treated H1R membrane (incubation time 30 min with the
phenoxybenzamine concentration at 90 nM) using IMAC purification system as previously
described in Chapter 3. Samples were collected at various stages of purification and they were
tested on the western blot and radioligand binding analysis. As shown in Figure 4B, we were able
to detect the protein band representing the H1R protein (~ 55 kDa) at every stages of purification.
On the other hands, when performing radioligand binding analysis on these collected samples, we
still observed the blockade of [ 3H]mepyramine binding on the H1R by phenoxybenzamine. These
results ensure the presence of H1R proteins and the irreversibly binding of phenoxybenzamine on
the H1R protein. The purified phenoxybenzamine-treated H1R protein were subjected to one
dimensional gel electrophoresis and stained with Coomassie Brilliant blue. The H1R protein bands
at 55 kDa were excised and analyzed by MALDI-ToF MS analysis. The experimentally determined
peptide masses from the phenoxybenzamine-treated and the non-treated H1R samples matched
well. We were also able to obtain a tryptic fragment from the phenoxybenzamine-treated H1R with
a mass difference corresponding to phenoxybenzamine (Figure 5).
Unexpectedly, the cysteine residue that was identified to be alkylated by phenoxybenzamine is at
the position 302 (Cys302) of the third intracellular loop of the H1R. Based on the structural insight
of the rhodopsin family of GPCRs (see Chapter 1 for further reading ), the binding pocket of the H1R
is thought to reside within the TM domains, such as TM3, TM5 and TM6. Furthermore, it has been
shown that the cysteine residue involved in the binding of phenoxybenzamine in the α-adrenergic
receptor is located at position 3.36 of TM3. We, therefore, expected to map the interaction of
phenoxybenzamine with amino acid residues inside the transmembrane domain region.
Interestingly, Espinoza-Fonseca and Trujillo-Ferrara have proposed in 2006 the binding of an
allosteric modulator, KT5720, to the intracellular region of the muscarinic M1 receptors (Espinoza-
Fonseca 2006) and 2 years later, Nicholls and co-workers have suggested the binding of the CXCR2
antagonist SB332235 to the intracellular region of CXCR2 (Nicholls 2008). It is tempting to
speculate that the H1R can also be modulated at the intracellular site, although it is also possible
that the covalent interaction between the Cys302 of the H1R and phenoxybenzamine from this
preliminary study is the result from a non-specific reaction, since the binding of phenoxybenzamine
to non-specific sites has also been observed previously (Terner 1971). Nevertheless, the mapping of
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MS analysis of the H1R: glycosylation and binding of an irreversible H1R antagonist
a cysteine residue in the H1R protein that is reactive to phenoxybenzamine provides evidence that
MS technology is definitely a promising proteomic tool in identifying amino acid residues involved
in ligand-protein interactions. For future MS analysis, the protocol regarding phenoxybenzamine
treatment and the H1R sample preparation can be adjusted to minimize potential non-specific
reactions. Furthermore, it is also noteworthy to further investigate the intracellular region of the
H1R as a possible binding site by mutation analysis together with modeling studies.
Figure 5. MALDI-ToF-MS analysis of the pre-treated H1R with phenoxybenzamine
A tryptic fragment from phenoxybenzamine-treated H1R was recovered and selected for MS/MS based on
the increase in the mass corresponding to phenoxybenzamine ( 268.2). The fragmentation pattern clearly
confirms that this peptide contains a phenoxybenzamine, most likely conjugated to the cysteine residue.
83 | P a g e

Chapter 4
84 | P a g e
Conclusion
The work presented here provides a promising MS analysis protocol that allows the
characterization of post-translational modifications (N-Linked glycosylation) and ligand binding
sites of the H1R. We succeeded for the first time in identifying a single amino acid residue (N5) in
the H1R that is glycosylated upon expression in insect cells. Furthermore, we also showed our
preliminary results in using the irreversible ligand phenoxybenzamine to identify direct ligand-H1R
protein interaction. Taken together, the use of affinity labels or other irreversible ligands together
with the MS technology will allow us to progress our studies on ligand-receptor interaction. The
precise knowledge of receptor bound ligand structures would provide opportunities for drug
discovery and aid the development of tailor-made medicines substantially.

CHAPTER
A structural insight into the reorientation
of transmembrane domains 3 and 5 during
H1R activation
Kamonchanok Sansuk 1 , Xavier Deupi 2 , Ivan Torrecillas 2 , Aldo Jongejan 1 ,
Remko Bakker 1, 3 , Leonardo Pardo 2 , Rob Leurs 1
1Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit Amsterdam,
Department of Medicinal Chemistry, De Boelelaan 1083, 1081HV, Amsterdam, the Netherlands
2Laboratori de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina,
Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain
3Current address: Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach, Germany
Jib
[Type the company name]
(This research is submitted to Journal of Molecular Pharmacology)
[Pick the date]

Chapter 5
86 | P a g e
Introduction
G protein-coupled receptors (GPCRs) transduce sensory signals of external origin such as photons,
odors or pheromones and endogenous signals, including neurotransmitters like histamine,
(neuro)peptides, proteases, glycoprotein hormones and ions, to the cytoplasmic side of the cell
membrane (Kristiansen 2004). The mechanism by which binding of these highly diverse chemical
signals triggers a set of conformational rearrangements of the transmembrane (TM) segments near
the G-protein binding domains remains largely unknown. NMR studies on the human histamine H1
receptor (H1R), a rhodopsin family type GPCR, using 13C, 15N-labelled histamine suggested that the
transition of the H1R from an predominantly inactive state to an active state after agonist binding
involves proton transfer, similar to the meta I/meta II transition equilibrium in rhodopsin (Ratnala
et al. 2007). Comparison of the structure of inactive rhodopsin (Li, Edwards et al. 2004) with the
recent crystal structure of the ligand-free opsin (Park, Scheerer et al. 2008), which contains several
distinctive features of the presumed active state, leads to the conclusion that during the process of
GPCR activation, among others changes, TM3 rotates clockwise (viewed from the intracellular side),
the intracellular part of TM6 tilts outwards by 6-7 Å, TM5 nears TM6, and R3.50 within the
(D/E)R(Y/W) motif in TM3 adopts an extended conformation pointing towards the protein core
(Park, Scheerer et al. 2008). Notably, as revealed in the original publication of opsin, these
conformational changes disrupt the ionic interaction between R3.50 with negatively charged side
chains at positions 3.49 and 6.30 (Smit, Vischer et al. 2007; Nygaard, Frimurer et al. 2009) and
facilitates the interactions between the highly conserved Y5.58 and K5.66 in TM5 and R3.50 in TM3
and E6.30 in TM6, respectively. This strongly suggests that both TM3 and TM5 play a central role in
stabilizing the active state of GPCRs and, therefore, in the process of GPCR activation. Previously,
we have reported that binding of histamine to the H1R initiates an interaction between TM3 and
TM7. Specifically, we found that S3.36 interacts with N7.49, freeing W6.48, which is otherwise fixed
in the inactive formation by N7.49. The S3.36-N7.49 serves as the first step in a cascade of other
transmembrane rearrangements that eventually lead to the receptor assuming the activated state,
in which it is able to activate its G-protein (Jongejan, Bruyster et al. 2005). In this study we set out
to further our knowledge of the activation of the H1R by combining the latest structural insights in
GPCR structure, computational modeling and site-directed mutagenesis of the H1R to study the role
of the TAS(I/V) motif at positions 3.37-3.40 in TM3, highly conserved in amine receptors, in the
process of receptor activation. Our results suggest that T3.37 interacts with TM5 in the inactive
state of the H1R, while S3.39 changes conformation upon receptor activation to achieve, together
with S3.36, new water-mediated inter-helical interactions with TM2 and TM7 that stabilize the

A structural insight into the reorientation of TM3 and TM5 during H1R activation
active state. In addition, we provide the first evidence that the hydrophobic side chain at position
3.40, highly conserved in the rhodopsin family of GPCRs, plays a key role in facilitating the
histamine-induced relocation of TM5. Mutation of I3.40 to either Ala or Gly, i.e. removing the bulky
side chain at this position, abolishes the constitutive activity of the histamine H1 receptor (H1R), the
effect of constitutive-activity increasing mutations, as well as the histamine-induced receptor
activation.
Materials
Experimental procedures
Gifts of mianserin hydrochloride (Schering-Plough, The Netherlands), pcDEF3 (Dr. J. Langer
(Goldman, Cutrone et al. 1996)) and of the cDNA encoding the human histamine H1R (Dr. H. Fukui
(Ohta, Hayashi et al. 1994)) are greatly acknowledged. pNF-κB-Luc was obtained from Stratagene
(La Jolla, USA), ATP disodium salt, bovine serum albumin, chloroquine diphosphate, DEAE-dextran
(chloride form), histamine dihydrochloride, mepyramine (pyrilamine maleate) and
polyethylenemine were purchased from Sigma Chemical Company (USA). D-luciferin was obtained
from Duchefa Biochemie BV (Haarlem, The Netherlands), glycerol from Riedel-de-Haen (Germany),
and Triton X-100 from Fluka (Switzerland). Cell culture media, penicillin and streptomycin were
obtained from Invitrogen Corporation (Paisley, UK). Fetal bovine serum was obtained from Intergro
B.V. (Dieren, The Netherlands). Cell culture plastics were obtained from Corning Costar (NY, USA).
[ 3H]mepyramine (30 Ci/mmol) was purchased-from ICN Biomedicals BV (Zoetermeer, The
Netherlands)
Cell culture and transfection
COS-7 African green monkey kidney cells were maintained at 37 °C in a humidified 5% CO2/95% air
atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 50 IU/ml penicillin, 50
µg/ml streptomycin and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected
using the DEAE-dextran method as previously described (Bakker, Schoonus et al. 2001).
Site-directed mutagenesis
Single point mutant human histamine H1Rs: T112A (T3.37A), T112E (T3.37E), I115A (I3.40A),
I115G (I3.40G), S114A (S3.39A), S114C (S3.39C) and S114T (S3.39T) and double mutant human
histamine H1Rs: S3.36T/I3.40A and S3.36T/I3.40G were created using a PCR-based mutagenesis
87 | P a g e

Chapter 5
approach (Cormack 2001). All constructs were sub-cloned into the expression vector pcDEF3 and
verified by DNA sequencing.
Reporter-gene assay
Reporter-gene assays were performed essentially as described previously (Bakker, Schoonus et al.
2001). In brief, cells transiently co-transfected with pNF-κB-Luc (125 µg/1.10 7 cells) and pcDEF3
encoding WT or mutant H1Rs (25 µg/1.10 7 cells) were seeded in 96 well white plates (Costar) in
serum free culture medium and incubated with drugs. After 48 hrs, cells were assayed for
luminescence by aspiration of the medium and the addition of 25 µl/well luciferase assay reagent
(0.83 mM ATP. 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 µM Na2H2P2O7, 38.9 mM Tris (pH 7.8).
0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100 and 2.6 µM dithiothreitol). After 30 min,
luminescence was measured for 3 sec/well in a Victor plate reader (Wallac).
Histamine H1R binding studies
Histamine H1R binding studies were performed essentially as described previously (Bakker,
Schoonus et al. 2001). In brief, transfected COS-7 cells used for radioligand binding studies were
harvested after 48hrs and homogenized in ice-cold 50 mM Na2/K-phosphate buffer (pH 7.4)
(binding buffer). The COS-7 cell homogenates were incubated for 30 min at 30 °C in binding buffer
in 200 µl with 3 nM [ 3H]mepyramine. Non-specific binding was determined in the presence of 1 µM
mianserin. The incubations were stopped by rapid dilution with 3 ml ice-cold binding buffer. The
bound radioactivity was separated by filtration through Whatman GF/C filters that had been
treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml binding buffer and
radioactivity retained on the filters was measured by liquid scintillation counting.
Analytical methods
Protein concentrations were determined according to Bradford (Bradford 1976), using BSA as a
standard. Binding and functional data were evaluated by a non-linear, least squares curve-fitting
procedure using GraphPad Prism 4 ® (GraphPad Software, Inc., San Diego, CA).
Computational model of the histamine H1R
A model of the TM domains 1-7 of the histamine H1R was build by homology modeling techniques
using the crystal structure of the β2AR (PDB code 2RH1) (Cherezov, Rosenbaum et al. 2007;
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A structural insight into the reorientation of TM3 and TM5 during H1R activation
Rasmussen, Choi et al. 2007) as template. Building this homology model was straightforward except
for the highly conserved NPxxYxn=5,6F motif at the junction between TM7 and the intracellular helix
8. Notably, this junction is one residue shorter in the β2AR (n=5) than in rhodopsin (n=6). As a
result, Y7.53 is pointing towards TM2 in rhodopsin (Li, Edwards et al. 2004) and towards TM6 in
the β2AR (Rasmussen, Choi et al. 2007). The fact that the histamine H1R contains six amino acids
(n=6) between both aromatic side chains led us to model the junction between TM7 and helix 8 as
in rhodopsin (PDB code 1GZM). SCWRL-3.0 (Canutescu, Shelenkov et al. 2003) was employed to
add the side chains of the non-conserved residues, using a backbone-dependent rotamer library.
Modeller 9v1 (Marti-Renom, Stuart et al. 2000) was used to add intracellular loops IL1-2 and
extracellular loops EL1-3 using the structure of the β2AR as template. Internal water molecules 506,
519, 528, 529, 532, 534, 537, 543, 546, and 548 that mediate a number of inter-helical interactions
(Rasmussen, Choi et al. 2007) and are probably conserved in the rhodopsin family of GPCRs
(Pardo, Deupi et al. 2007) were explicitly included in the model.
Distortion of TM5
The program HELANAL (Bansal, Kumar et al. 2000) was used to calculate the residue-residue unit
twist angle of TM5, a structural parameter that describes local helical uniformity. This parameter is
interpreted as follows: an ideal α-helix, with approximately 3.6 residues per turn, has a twist angle
of approximately 100º (360º/3.6); a closed helical segment, with 100º; whereas an open helical segment, with >3.6 residues per turn, possesses a twist

Chapter 5
receptor the constitutive activity decreases (right column). On the contrary, a mutation that
decreases the strength of an intramolecular interaction (i.e., Ser to Ala, Asp to Asn, …) (Table 1A,
bottom row) leads to an increase (left column) or a decrease (right column) of the constitutive
activity depending whether the mutated residue is stabilizing the inactive or the active state of the
receptor, respectively. In addition, single point mutations that modify the side chain rotamer
distribution, without modifying its capability to form intramolecular interactions, can help to
elucidate the conformer involved in the stabilization of either the inactive or active state of the
receptor (Table 1B).
Analysis of structural databases shows that Ser, Thr, and Cys residues possess different side chain
rotamer distribution when located in a �-helical structure (Ballesteros, Deupi et al. 2000). Ser can
adopt either the g+ (52% of the side chains), g- (20%), or t (28%) rotamer conformation; the β-
branched side chain of Thr is limited to the g+ (85%) and g- (15%) because the t conformation is
unfavorable due to the steric clash of the side chain methyl group with the backbone carbonyl at the
i-3 position; while Cys is restricted to the g+ (71%) or t (29%) conformation because of the steric
clash between the Sγ atom and the carbonyl oxygen of residue i-3 in the g- conformation
(McGregor, Islam et al. 1987). Thus, if mutation of Ser (able to adopt the g+, g- or t conformations)
to Cys (g+ and t) (Table 1B, top row) and Thr (g+ and g-) (Table 1B, bottom row) increases, in both
cases, the constitutive activity (left column), Ser adopts the g+ conformation in the active state, as
both mutations lead to an increase of the relative population of the g+ conformation. Conversely, if
these mutations lead to a decrease of the constitutive activity (right column), the g+ conformation
of Ser would stabilize the inactive conformation of the receptor.
Numbering Scheme of GPCRs
Residues are identified by the generic numbering scheme of Ballesteros & Weinstein (Ballesteros
and Weinstein 1995) that allows easy comparison among residues in the 7TM segments of different
receptors.
Sequence analysis
Family-specific analysis of GPCR sequences were performed using the GMoS program, freely
available at http://lmc.uab.cat/gmos.
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A structural insight into the reorientation of TM3 and TM5 during H1R activation
Table 1 Correspondence between single point mutations, experimentally determined constitutive
agonist-independent activity, and receptor structure
Combination of structural bioinformatics with experimental results allows deducing the
structural/functional role of the mutated residues.
STRUCTURAL BIOINFORMATICS
A
B
Effect of the
mutation on the
strength of the
intramolecular
interaction
Effect of the
mutation on the
side chain
conformation
distribution
increase strength
(e.g. Ala → Ser)
decrease strength
(e.g. Ser → Ala)
Ser → Cys
g+/g-/t g+/t
Ser → Thr
g+/g-/t g+/g-
EXPERIMENTAL RESULTS
Constitutive activity (CA)
increase CA decrease CA
Side chain
stabilizes the
ACTIVE
conformation
Side chain
stabilizes the
INACTIVE
conformation
Ser in g+
stabilizes the
ACTIVE
conformation
Side chain
stabilizes the
INACTIVE
conformation
Side chain
stabilizes the
ACTIVE
conformation
Ser in g+
stabilizes the
INACTIVE
conformation
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Results
A proline-induced unwinding in the structure of TM5 of Class A GPCRs.
GPCRs, including the H1R, possess a highly conserved P5.50 in TM5. This conserved proline
presents in 77% of the rhodopsin-like sequences (Mirzadegan, Benko et al. 2003), and absents only
in melanocortin, glycoprotein hormone, lysosphingolipid, prostanoid, and cannabinoid receptors.
Usually, in Pro-containing α-helices, the steric clash between the pyrrolidine ring of Pro and the
backbone carbonyl oxygen in the preceding turn induces a bend angle of approximately 20º in the
helical structure (Deupi, Olivella et al. 2004). In contrast, the structures of bovine (Li, Edwards et al.
2004) and squid (Murakami and Kouyama 2008) rhodopsin, adenosine A2A receptor (Jaakola,
Griffith et al. 2008), and the β1AR (Warne, Serrano-Vega et al. 2008) and β2AR (Cherezov,
Rosenbaum et al. 2007; Rasmussen, Choi et al. 2007) have revealed a peculiar conformation of TM5
in which the Pro-kink induced bend is decreased (Figure 1A) due to a local opening of the helix
(>3.6 residues/turn, twist < 100º, see Experimental Procedures) at the 5.45-5.48 turn (Figure 1B)
that partially removes the steric clash between the pyrrolidine ring of P 5.50 and the backbone
carbonyl oxygen at position 5.46 (i-4 in Figure 1C and 1D). This local opening of TM5 (proline-
unwinding, in contrast to proline-kink) also modifies the relative orientation of the side chains at the
extracellular side (Govaerts, Lefort et al. 2001; Deupi, Dolker et al. 2007), including residues
involved in neurotransmitter binding such as 5.42, 5.43, and 5.46 (Shi, Liapakis et al. 2002). Other
membrane proteins also feature this type of helical wide turns (Riek, Finch et al. 2008).
The role of TM3 in stabilizing the proline-unwinding of TM5
The mechanism by which this unusual conformation of TM5 is stabilized remains unknown. In
bovine rhodopsin, the backbone carbonyl oxygen at position 5.46 is stabilized by a hydrogen bond
interaction with E3.37 (at the inter-atomic distance of 2.8Å) and a van der Waals interaction with
L3.40 (3.2Å) (Figure 1C) (Li, Edwards et al. 2004; Deupi, Dolker et al. 2007). Adenosine A2A
receptor preserves similar interactions through Q3.37 (3.2Å) and I3.40 (3.9Å) (Jaakola, Griffith et
al. 2008). Interestingly, squid rhodopsin replaces E3.37 of bovine rhodopsin by Phe but presents a
discrete water molecule linking the backbone carbonyls at positions 3.37 (3.0Å) and 5.46 (3.3Å)
(Murakami and Kouyama 2008), while maintaining the van der Waals interaction between the
hydrophobic I3.40 and the carbonyl oxygen at position 5.46 (3.2Å). Notably, the recent X-ray
structure of opsin (Park, Scheerer et al. 2008) shows that during the process of GPCR activation the
hydrogen bond interaction between E3.37 and the backbone carbonyl (4.9Å) is disrupted, as

A structural insight into the reorientation of TM3 and TM5 during H1R activation
previously suggested by NMR measurements of rhodopsin containing 13C-labeled histidine (Patel,
Crocker et al. 2005), while L3.40 has moved slightly away (3.5Å) (Figure 1D).
This effect correlates with the minor, but significant, clockwise rotation (viewed from the
intracellular side) of TM3 during the process of receptor activation (see Discussion). The release of
the strong packing of L3.40 with the 5.45-5.48 turn does not result in a change in the local opening
of TM5 (Figure 1B), but in a localized decrease in the bend around P5.50 (Figure 1A). In the β1AR
(Warne, Serrano-Vega et al. 2008) and β2AR (Cherezov, Rosenbaum et al. 2007; Rasmussen, Choi et
al. 2007), I3.40 also stabilizes the carbonyl oxygen at position 5.46 (i-4 in Figure 1E), similarly to
adenosine and opsin receptors, whereas, in this case, the shorter T3.37 (compared to E or Q)
interacts with the side chain, rather than with the backbone carbonyl, of S5.46. To investigate the
role of the amino acids at positions 3.37 and 3.40 in GPCR activation we have created and studied
several histamine H1 receptor (H1R) mutants. We have previously used the H1R as a model system
for the study of Class A GPCR activation (Bakker, Jongejan et al. 2008). The H1R belongs to the
aminergic subfamily of Class A GPCRs, and like the β1AR and β2AR features T3.37 and I3.40 in TM3.
Moreover, the H1R contains a polar N5.46 amino acid at position 5.46, which is involved in
histamine binding (Leurs, Smit et al. 1994), linking this GPCR sub-domain directly to the agonist
binding site.
Influence of T3.37 and I3.40 in agonist-induced activation of the human histamine H1R
In order to test the role of the amino acids at positions 3.37 and 3.40 we engineered mutants in
which T3.37 of the histamine H1R was substituted by either Ala (T3.37A), to remove the hydrogen
bonding capability at this position, or Glu (T3.37E), to mimic the bovine rhodopsin sequence; and
I3.40 was replaced by either Ala (I3.40A) or Gly (I3.40G) removing the hydrophobic and bulky side
chain and, thus, the van der Waals interaction with the carbonyl group at position 5.46. Figure 2E
shows the environment of T3.37 in the β2-based molecular model of the histamine H1R (see
Experimental Procedures). T3.37 hydrogen bonds N5.46, the binding partner for the imidazole ring
of histamine (Leurs, Smit et al. 1994), in a similar manner as the T3.37•••S5.46 interaction in the
structure of the β2AR (Figure 1E). Wild type, T3.37A and T3.37E mutants H1Rs were well expressed
in COS-7 cells (Bmax 14.8±4.6 (n=7), 8.5±2.1 (n=2) and 12.8±3.3 (n=2) pmol/mg protein,
respectively) as determined by radioligand binding analysis (Table 2).
93 | P a g e

Chapter 5
The function of the WT H1R was evaluated in a NF-κB reporter-gene assay as previously reported
(Jongejan, Bruysters et al. 2005; Bakker, Jongejan et al. 2008), and the activation of NF-κB was
increased up to 10 fold when stimulated with histamine (pEC50=6.5±0.1) (Table 2 and Figure 2A).
Removal of the interaction of T3.37 with N5.46, by mutating residue T3.37 to Ala resulted in a 4-
fold decrease of the binding affinity of histamine (pKi of 5.3 for the WT H1R vs. 4.7 for the T3.37A
H1R) (Table 2) as measured by [ 3H]mepyramine displacement. A lower constitutive signaling of the
T3.37A H1R mutant compared to that of WT H1R was observed (Figure 2A and 2B). However, the
basal signaling could still be increased by histamine albeit at higher concentrations (pEC50=4.5±0.2)
(Table 2).
Mutation of residue T3.37 to Glu (i.e. making this region similar to bovine rhodopsin) did not
influence the binding affinity of histamine (pKi of 5.3 for the WT H1R versus 5.1 for the T3.37E H1R),
possibly because E3.37 maintains both the interaction with N5.46 (Figure 2F), as in the WT H1R
(Figure 2E), and with the carbonyl group at position 5.46, as in bovine rhodopsin (Figure 1C).
Notably, the T3.37E mutant H1R hardly shows any constitutive activity (Figure 2A and 2B).
Moreover, the T3.37E mutant H1R is activated very poorly by histamine (pEC50>3.5) (Table 2).
Thus, we propose that the additional constraint between E3.37 and the backbone carbonyl at
position 5.46, introduced in the T3.37E mutation, impedes the activation of the mutant H1R by
locking a local structure that cannot be overcome by the agonist histamine. Our results also show
that removal of the bulky hydrophobic I3.40 side chain by substitution to Ala or Gly is translated
into significant effects on both the constitutive activity of the receptor and the process of histamine-
induced receptor activation (Figure 2A and 2C). Importantly, these effects are not due to a loss of
binding affinity for histamine. As shown in Table 2 both I3.40A and I3.40G mutant H1Rs are well
expressed in COS-7 cells (Bmax 9.4±5.1 and 8.8±4.8 pmol/mg protein, respectively) and bind
histamine with comparable affinity (pKi = 5.3±0.2 for I3.40A H1R and 5.0±0.2 for the I3.40G H1R)
compared to WT H1R (pKi = 5.3±0.1).
The fact that removal of the I3.40 side chain abolished both the constitutive and histamine-induced
GPCR activity strongly suggests that this hydrophobic side chain plays a key role in the mechanism
of activation. Based on these results and on the recent insights in GPCR structure (Park, Scheerer et
al. 2008; Scheerer, Park et al. 2008), we propose that the hydrophobic side chain at position 3.40,
which is strongly packed against TM5 (Figure 1), acts as a pivot and the Pro5.50-induced
unwinding acts as a hinge in the crucial reorientation of TM5 upon activation, which ultimately
94 | P a g e

A structural insight into the reorientation of TM3 and TM5 during H1R activation
facilitates the interactions of Y5.58 and K5.66 with R3.50 and E6.30, respectively (Figure 1).
Notably, mutations at position 3.40 in rhodopsin are related to retinitis pigmentosa, poor retinal
binding, misfolding, reduced expression, decreased time in the MII state, and reduced transducin
activation (Madabushi, Gross et al. 2004).
Table 2 Pharmacological characteristics of hH1R wild type (WT) and H1R mutants transiently
expressed in COS-7 cells.
The potency (pEC50) of histamine was measured by NF-κB reporter gene assay. Affinities of [ 3 H]mepyramine
(KD) and expression levels are determined by saturation radioligand binding assays. Binding affinity values of
H1R ligands are determined by [ 3 H]mepyramine displacement. Data were calculated as the mean ± S.E.M. (n)
is number of experiments, each performed in triplicate.
Receptor
KD (nM)± S.E.M.
(n)
[ 3H]mepyramine Histamine
Bmax(pmol/mg
protein)
± S.E.M. (n)
pKi± S.E.M. (n) pEC50± S.E.M. (n)
hH1R WT 1.5 ± 0.2 (7) 14.8 ± 4.6 (7) 5.3 ± 0.1 (4) 6.5 ± 0.1 (5)
T3.37A 4.8 ± 0.3 (2) 8.5 ± 2.1 (2) 4.7 ± 0.1 (4) 4.5 ± 0.2 (3)
T3.37E 6.2 ± 0.1 (2) 12.8 ± 3.3 (2) 5.1 ± 0.1 (4) > 3.2 (3)
S3.39A 1.6 ± 0.3 (3) 8.9 ± 2.4 (3) 5.4 ± 0.1 (4) 4.9 ± 0.1 (3)
S3.39C 1.5 ± 0.3 (3) 9.0 ± 3.2 (3) 5.3 ± 0.1 (4) 5.2 ± 0.1 (3)
S3.39T 3.6 ± 0.3 (3) 8.2 ± 4.7 (3) 5.4 ± 0.1 (4) 4.9 ± 0.2 (3)
I3.40A 10.1 ± 0.3 (3) 9.4 ± 5.1 (3) 5.3 ± 0.2 (4) > 3.4 (3)
I3.40G 7.5 ± 0.2 (2) 8.8 ± 4.8 (2) 5.0 ± 0.2 (3) > 3.8 (2)
S3.36T+
I3.40A
S3.36T+
I3.40G
7.8 ± 0.3 (4) 10.4 ± 5.3 (4) 5.4 ± 0.1 (3) > 3.7 (2)
3.4 ± 0.2 (3) 13.4 ± 5.0 (3) 5.0 ± 0.1 (3) > 3.9 (2)
To substantiate this hypothesis we performed an additional set of experiments. We have recently
shown that the S3.36T mutation in the histamine H1R induces a large increase in constitutive
activity (Jongejan, Bruysters et al. 2005). We proposed that T3.36 induces the transition of W6.48
toward TM5 (Jongejan, Bruysters et al. 2005; Pellissier, Sallander et al. 2009), considered to be the
initial stage of the activation process, as observed in the electron microscopy density map of
95 | P a g e

Chapter 5
metarhodopsin I (Ruprecht, Mielke et al. 2004) and in solid-state NMR measurements of
metarhodopsin II (Crocker, Eilers et al. 2006). Thus, we combined the activating S3.36T point
mutation with the inactivating I3.40A/G mutation to generate the S3.36T/I3.40A and
S3.36T/I3.40G double mutant H1Rs, with the aim to assess their compensatory consequences on
receptor function. The double mutant H1Rs are well expressed in COS-7 cells (Bmax 10.4±5.3 and
13.4±0.3 pmol/mg protein, respectively) and have similar affinity for histamine compared to wild-
type H1R (pKi 5.4±0.1 for the S3.36T/I3.40A H1R and 5.0±0.1 for the S3.36T/I3.40G H1R,
respectively) (Table 2).
Interestingly, both double mutants show lack of constitutive activity and cannot be activated by
histamine, resembling the single I3.40A/G mutant H1R (Figure 2A and 2C). These data reinforce
the importance of I3.40 in the process of receptor activation.
The role of S3.36 and S3.39 in the process of histamine H1R activation
S3.39, within the TAS(I/V) motif, is highly conserved in the biogenic amine (100% of the
sequences) and other (S:75%; T:6%) GPCR families, which suggests a conserved structural and/or
functional role. S3.39, absent in bovine rhodopsin, is hydrogen bonding, in the g+ conformation, the
backbone carbonyl at position 3.35 in the previous turn of the helix in the structures of β1AR (at the
inter-atomic distance between heteroatoms of 3.0Å) and β2AR (2.7Å) adrenergic receptors and
adenosine A2A receptor (3.1Å). S3.39 does not interact with the near and highly conserved D2.50 in
TM2 (4.7Å, 4.1Å and 4.6Å in the β1AR, β2AR and A2A receptors, respectively) or near water
molecules.
Thus, to study the role of this amino acid on the receptor structure and function (see Table 1) we
mutated S3.39 to alanine, threonine and cysteine. The S3.39A mutant H1R is well expressed in COS-
7 cells (Bmax 8.9±2.4 pmol/mg protein (n=3)) and has a similar binding affinity for histamine (pKi
5.4±0.1 (n=4)) compared to the wild-type H1R (Table 2). As shown in the NF-κB reporter gene
assay (see Experimental Procedures), the S3.39A mutant H1R shows almost no constitutive activity
(Figure 3A). However, the S3.39A mutant H1R could still be activated by histamine with pEC50 of
4.9±0.1 (n=3), but the maximal activity is reduced by a factor of 2 (Figure 3A and 3B). These
results indicate that the hydrogen bond capability of S3.39 plays an important role in stabilizing the
active state of the histamine H1R. Furthermore, S3.39T/C mutations provide a way of identifying
96 | P a g e

A structural insight into the reorientation of TM3 and TM5 during H1R activation
the conformation of S3.39 in the active state because of the different side chain rotamer
distribution of serine from those of threonine or cysteine, when located in an α-helical structure
(see Experimental Procedures, Table 1).
Figure 1 The local opening of transmembrane helix 5 in bovine rhodopsin (PDB code 1GZM),
opsin (3CAP) and the β2-adrenergic receptor (2RH1)
(A, B) Evolution of bend (A) and helical twist (B) angles (°, see Experimental Procedures) along TM5 in the
crystal structures of the β2-adrenergic receptor (blue), bovine rhodopsin (black) and opsin (red). Residue
number refers to the first residue in each turn; i.e. the peak of the helical distortion appears in the turn 5.45-
5.48, labeled as 5.45 in the graphic. (C, D, E) Detailed view of the interface between TMs 3 (dark red) and 5
(green) in bovine rhodopsin (C), opsin (D), and the �2-adrenergic receptor (E) in views parallel (top) and
perpendicular (bottom) to the membrane.
97 | P a g e

Chapter 5
Furthermore, both S3.39T/C mutant H1Rs also lack constitutive activity and their maximal activites
are also reduced by a factor of 2 relative to WT (Figure 3A and 3B). These results can be explained
by an increase of the population of the g+ side chain conformation, which corresponds to the
inactive state (see above). These findings suggest that the side chain at position 3.39 changes from
the inactive g+ to either the g- or t active conformation upon activation. In Class A GPCRs position
3.36 is highly variable; however, in amine receptors it is mostly C (57%) or S (32%), with the
notable exception of β-adrenergic receptors, which feature V. Remarkably, T is virtually absent
(2%). The S3.36T mutation showed a large increase in constitutive activity (Figure 2A and 2D),
suggesting that the side chain at this position plays a key role in stabilizing the active state of the
receptor in the g+ conformation, pointing towards TM7 (Jongejan, Bruysters et al. 2005).
This conformational transition of S3.36 and the rotation of TM3 are only feasible if the side chain of
W6.48 modifies its conformation simultaneously, in a concerted manner, from the inactive g+
(pointing towards TM7) to the active t conformation (pointing towards TM5) (Jongejan, Bruysters
et al. 2005; Pellissier, Sallander et al. 2009). Figure 3C and 3D depict the conformational transition
of these side chains and the proposed interactions in the inactive and active states.
The rotamer toggle switch of W6.48 relocates the indole ring of the side chain from pointing
towards TM7, where it is engaged in a conserved, water-mediated, hydrogen bond interaction with
D2.50 (Figure 3C) (Pardo, Deupi et al. 2007), to point towards the binding site crevice (Figure 3D)
(Pardo, Deupi et al. 2007; Pellissier, Sallander et al. 2009).
The rotamer toggle switch of S3.36 relocates its polar side chain from the binding pocket (Figure
3C) (Jongejan, Bruysters et al. 2005) to point towards TM7 where it might fulfill a hydrogen bond
interaction with the water molecules in the D2.50/W6.48 environment (Figure 3D). Finally, S3.39
also switches from the inactive g+, hydrogen bonding the backbone carbonyl in the previous turn of
the helix (Figure 3C), to point towards TM7 where it stabilizes the active state of the receptor by
interacting with the water molecules in the D2.50/W6.48 environment (Figure 3D).
98 | P a g e
DISCUSSION
A number of studies have provided convincing evidence that GPCRs co-exist in different
conformations (Kobilka and Deupi 2007). GPCRs are maintained within the ensemble of inactive

A structural insight into the reorientation of TM3 and TM5 during H1R activation
conformations through non-covalent interactions between side chains, mostly located in the TM
segments (Smit, Vischer et al. 2007; Weis and Kobilka 2008; Nygaard, Frimurer et al. 2009).
Disruption of these stabilizing interactions by Ala-scanning mutagenesis leads to gain-of-function
mutations. The crystal structures of the inactive state of bovine (Li, Edwards et al. 2004) and squid
(Murakami and Kouyama 2008) rhodopsin, adenosine A2A receptor (Jaakola, Griffith et al. 2008),
and the β1AR (Warne, Serrano-Vega et al. 2008) and β2AR (Cherezov, Rosenbaum et al. 2007;
Rasmussen, Choi et al. 2007) facilitate the structural interpretation of these experimental results.
However, understanding the mechanisms that shift the equilibrium of the ensemble to the active
conformations is more challenging for two reasons. First, alanine-scanning mutagenesis of the side
chains involved in the stabilization of active conformations leads to loss-of-function effects (Table
1), which are more difficult to measure with precision. Second, high-resolution structural
information of the active state is very scarce, currently limited to the cytoplasmic side of the recent
crystal structure of the ligand-free opsin (Park, Scheerer et al. 2008), which hampers the structural
interpretation of the experimental results. The fact that the histamine H1R shows considerable
agonist-independent, constitutive signaling, which can be strongly reduced by the inverse agonist
mepyramine (Bakker, Schoonus et al. 2001), provides an unique opportunity to approach the
mechanism of receptor activation. We have designed point mutations that modulate the strenght of
intramolecular interactions and the side chain rotamer distribution of specific residues, and
measured their effect on constitutive agonist-independent activity (see Table 1).
Specifically, we have focused on the highly conserved TAS(I/V) motif in TM3 of amine receptors. An
evolutionary trace analysis has led to the identification of a putative common signal transduction
process for Class A GPCRs, where key residues are grouped into a network of interactions extending
from the binding pocket to the cytoplasmic region (Madabushi, Gross et al. 2004). This analysis
detected L3.40 of rhodopsin as a structurally and/or functionally important residue.
Our results support a role of this amino acid in the process of histamine H1R activation. Mutation of
I3.40 to either Ala or Gly, removing the bulky side chain, abolished the constitutive activity of the
receptor, the effect of constitutive-activity increasing mutations, and the histamine-induced
receptor activation. In the inactive state (Figure 1C and 1E) this hydrophobic side chain at position
3.40 (green surface) is located between the pyrrolidine ring of P5.50 and the carbonyl oxygen at
position 5.46, inducing or stabilizing the local opening of TM5 (Figure 1B).
99 | P a g e

Chapter 5
Figure 2 Influence of T3.37 and I3.40 in the agonist-induced activation of the histamine H1
receptor. NF-κB activation modulated by histamine in COS-7 cells transiently transfected with
wild type (WT), T3.37, I3.40, S3.36, double H1R mutants and empty vector (pcDEF3)
(A) Basal activity (black bars) and after stimulation with 10-4 M histamine (grey bars). Results are normalized to
the basal activity of WT which is put as 100%. (B) Representative dose response curves of histamine for WT (●),
T3.37A (○), T3.37E (�), and pcDEF3 (□). (C) Representative dose response curves of histamine for WT (●), I3.40A
(○), I3.40G (�), and pcDEF3 (□). (D) Representative dose response curves of histamine for WT (●), S3.36T (○),
S3.36T+I3.40G (�), S3.36T+I3.40A (♦) and pcDEF3 (□). (E, F) Detailed view of the interface between TMs 3 and 5
in the molecular model of WT (E) and mutant T3.37E (F) histamine H1R.
100 | P a g e

A
B
C
D
Figure 3 Influence of
S3.39 in the agonistinduced
activation of
the histamine H1
receptor
(A) Basal activity (black
bars) and after stimulation
with 10 -4 M histamine (grey
bars). Results are
normalized to the basal
activity of WT which is put
as 100%. (B)
Representative dose
response curves of
histamine for WT (•),
S3.39T (ο), S3.39A (�),
S3.39C ( ) hH1R mutants
and pcDEF3 (♦). (C, D)
Proposed hydrogen-bond
network involving the side
chains of D2.50, S3.36,
S3.39 and W6.48, and the
water molecules in the
D2.50/W6.48 environment
in the inactive (C) and
active (D) models of the
histamine H1R.

Chapter 5
However, the molecular mechanism by which this residue facilitates receptor activation is not
straightforward. The structure of opsin (Park, Scheerer et al. 2008) and previous substituted-
cysteine accessibility studies in the β2AR (Gether, Lin et al. 1997) and µ- opioid receptor(Shi,
Liapakis et al. 2002) have shown a clockwise rotation (viewed from the intracellular side) of TM3
during the process of receptor activation. Rotation of TM3 moves the bulky side chain at position
3.40, located between P5.50 and the 5.46 carbonyl, away from these moieties (Figure 1D), resulting
in a localized decrease of the bend around P5.50 (Figure 1A).
Fluorescence spectroscopy experiments used to monitor agonist-induced conformational changes
in the β2AR provide evidence of a sequential binding model through discrete intermediate
conformational states (Yao, Parnot et al. 2006). In an initial step agonist binding induces relocation
of the extracellular side of TM5 to facilitate binding of the catechol hydroxyls of the β2AR ligand
(Kobilka 2004). We propose that this movement is transduced as a set of structural rearrangements
toward the intracellular side of TM5 with the assistance of I3.40 acting as a pivot (Figure 2C),
ultimately enabling the interactions of Y5.58 with R3.50 and K5.66 with E6.30 in the cytoplasmic
side of the receptor.
In support of this hypothesis, the T3.37E mutation provides an additional constraint between TMs
3 and 5 (Figure 2F), thereby preventing the transmission of the activation signal through TM5
(Figure 2B). In addition to this activation pathway through TM5, agonist binding triggers
additional routes of activation, for instance by activating the rotamer toggle switch of tryptophan at
position 6.48 (W6.48) from the inactive g+ (pointing towards TM7) to the active t (towards the
binding pocket) conformation (Shi, Liapakis et al. 2002; Ruprecht, Mielke et al. 2004; Crocker, Eilers
et al. 2006; Ahuja, Crocker et al. 2009) (Figure 3D and 3E), through the formation of specific
hydrogen bonds (Pardo, Deupi et al. 2007; Pellissier, Sallander et al. 2009).
Notably, the rotamer toggle switch of W6.48 occurs in a concerted manner with the side chain at
position 3.36 from the inactive t (towards the binding pocket) to the active g+ (towards TM7)
conformation (Figure 3D and 3E) (Jongejan, Bruysters et al. 2005; Pellissier, Sallander et al. 2009).
In this manuscript we have first shown that S3.39 also switches from the inactive g+ (hydrogen
bonding the backbone carbonyl in the previous turn of the helix) to the active g- or t conformer,
pointing towards TM7 and stabilizing the active state of the receptor probably by interacting with
the water molecules in the D2.50/W6.48 environment (Figure 3E).
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A structural insight into the reorientation of TM3 and TM5 during H1R activation
In the H1R, histamine binding induces and/or stabilizes the conformational switches of S3.36, S3.39,
and W6.48 (toggle switch), triggering the conformational transition of N7.49 (the NPxxY motif of
TM7) towards D2.50 (Urizar, Claeysen et al. 2005) (Figure 4), and ultimately lead to the disruption
of the ionic interaction between R3.50 and the adjacent D/E3.49 (the DRY motif of TM3) (Scheer,
Fanelli et al. 1996), and an additional D/E6.30 near the cytoplasmic end of TM6 (Govaerts, Lefort et
al. 2001) that is not present in several GPCR families (Springael, de Poorter et al. 2007). Disruption
of these ionic interactions permit R3.50 to adopt an extended conformation (Bakker, Jongejan et al.
2008; Park, Scheerer et al. 2008), pointing toward the protein core, to interact with the highly
conserved Y5.58 and Y7.53 in TMs 5 and 7 (Park, Scheerer et al. 2008).
A recent computer model analysis of the activation mechanism of the guinea pig H1R found
somewhat similar findings. The model proposes that during the first stage of receptor activation,
straightening of TM VI, causes a rearrangment bulky aminoacids in TM VI. This then causes the
interaction between of S3.39 that interacts with N7.45 to be lost. After activation S3.39 moves to
TM2 to form a water mediated hydrogen bond with D2.50. Unlike the human version, in the guinea
pig H1R there is only a very limited role for S3.36 in the activation process (Strasser, Wittmann et al.
2007).
Thus, there is an activation pathway leading from agonist binding to the cytoplasmic side of the
receptor through TMs 3, 6 and 7 (Figure 4, left arrows). Fluorescence spectroscopy in the β2AR has
shown that partial agonists are as effective as full agonists in disrupting the ionic interaction of
R3.50, therefore, additional pathways of signal propagation must exist (Yao, Parnot et al. 2006). We
propose that one of such pathways consists in the agonist-induced relocation of the extracellular
side of TM5 towards the bundle, which is transmitted towards the cytoplasmic region through the
fulcrum/lever mechanism described above, in which P5.50 acts as a hinge and the highly conserved
hydrophobic I3.40 side chain acts as a pivot (Figure 4, right arrow). Triggering specific pathways
along the activation process might be an explanation for the observed collateral efficacy (Kenakin
2007) of GPCR agonists. A thorough understanding how a ligand-encoded extracellular signal is
propagated from its binding site into intracellular micro-domains will have a big impact on our
understanding of the physiology and pathology of this important family of proteins, and in the
therapeutic use of agonists and GPCR modulators in the clinic.
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Chapter 5
Figure 4 Pathways of activation in the rhodopsin family of GPCRs
Agonist binding at the extracellular domain of the receptor leads to an activation pathway through TMs 3, 6
and 7 (left arrows); or through relocation of TM5 assisted by the conserved hydrophobic side chain at
position 3.40, depicted as a green sphere (right arrow).
104 | P a g e

CHAPTER
The role of isoleucine at position 6.45 in
the H1R
Kamonchanok Sansuk 1, Remko Bakker 1, 3, Anne Watts 1, Leonardo
Pardo 2, Rob Leurs 1
1Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit Amsterdam,
Department of Medicinal Chemistry, De Boelelaan 1083, 1081HV, Amsterdam, the Netherlands
2Laboratori de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina,
Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain
3Current address: Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach, Germany
Jib
[Type the company name]
[Pick the date]

Chapter 6
106 | P a g e
Introduction
Several mediators are involved in the patho-physiology of allergic diseases. However, histamine,
which is synthesized and released by human basophils, mast cells, lymphocytes, neurons and
gastric enterochromaffin-like cells, remains the principal molecule that plays a fundamental role in
the genesis of these diseases, particularly rhinitis and urticaria. To date four subtypes of histamine
receptors have been described (H1, H2, H3 and H4 receptors), which all belong to the super family of
G-protein coupled receptors (GPCRs). Histamine is thought to exert its effects in allergic disease
primarily through the interaction with the histamine H1 receptor (H1R), which is present in a
variety of cells (Leurs, Church et al. 2002; Akdis and Simons 2006). Consequently, the H1R is an
important drug target, as during the past 20 years H1-receptor antagonists have become one of the
most frequently prescribed medications in Western countries to relieve the symptoms of allergic
reactions (Pierce, Premont et al. 2002; Lundstrom 2005). The H1R exists in equilibrium between its
inactive and active state. Histamine and other H1R agonists stabilize the active conformation and
thereby promote H1R-mediated signal transduction. Even in the absence of H1R agonists the H1R
can also be spontaneously active (Bakker, Schoonus et al. 2001). The constitutive activity of the H1R
can be inhibited by H1R inverse agonists like mepyramine that stabilize the inactive conformation
of the receptor and thereby block H1R-mediated signal transduction (Leurs, Church et al. 2002).
Constitutive activity may also be induced by mutations of amino acid residues in the receptor.
These constitutively active mutants have been successfully used for several GPCRs to gain insight in
the mechanism of receptor activation (Ballesteros, Jensen et al. 2001; Govaerts, Lefort et al. 2001;
Jongejan, Bruysters et al. 2005).
In order to create constitutively active mutant H1R, we previously employed random mutagenesis
in transmembrane domain (TM) regions known to be important for the receptor activation (Bakker,
Jongejan et al. 2008). These regions included the highly conserved DRY motif in TM3, the top of
TM5 and of TM6. The subsequently generated mutants were initially screened using the receptor
selection and amplification technology (R-SAT) and three residues in TM6: I420 (I6.40), A423
(A6.43) and I425 (I6.45) were identified as sites prone to yield highly constitutively active mutants
(Bakker, Jongejan et al. 2008). Further characterization of these constitutively active mutants in a
nuclear factor-κ B (NF-κB) reporter gene assay led to the finding that the I6.45R H1R mutant
exhibits an exceptional pharmacological profile. Mutation of H1R at position 6.45 from isoleucine to
arginine resulted in a unique receptor that no longer gives a NF-κB response when stimulated with

The role of isoleucine at position 6.45 in the H1R
the endogenous ligand histamine. In this study, we therefore pharmacologically characterized the
I6.45R H1R mutant in detail in order to gain insights in the mechanism of the H1R activation.
Materials
Experimental procedures
Gifts of mianserin hydrochloride (Schering-Plough, the Netherlands), pcDEF3 (Dr. J. Langer
(Goldman, Cutrone et al. 1996)) and of the cDNA encoding the human histamine H1R (Dr. H. Fukui
(Ohta, Hayashi et al. 1994)) are greatly acknowledged). All H1R mutants are available in the lab
(Bakker, Jongejan et al. 2008). pNF-κB-Luc was obtained from Stratagene (La Jolla, USA), ATP
disodium salt, bovine serum albumin, chloroquine diphosphate, DEAE-dextran (chloride form),
histamine dihydrochloride, mepyramine (pyrilamine maleate) and polyethylenemine were
purchased from Sigma Chemical Company (USA). D-luciferin was obtained from Duchefa Biochemie
BV (Haarlem, The Netherlands), glycerol from Riedel-de-Haen (Germany), and Triton X-100 from
Fluka (Switzerland). Cell culture media, penicillin and streptomycin were obtained from Invitrogen
Corporation (Paisley, UK). Fetal bovine serum was obtained from Intergro B.V. (Dieren, The
Netherlands). Cell culture plastics were obtained from Corning Costar (NY, USA). [ 3H]mepyramine
(30 Ci/mmol) was purchased-from ICN Biomedicals BV (Zoetermeer, The Netherlands)
Cell culture and transfection
COS-7 African green monkey kidney cells were maintained at 37 °C in a humidified 5% CO2/95% air
atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 50 IU/ml penicillin, 50
µg/ml streptomycin and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected
using the DEAE-dextran method as previously described (Bakker, Schoonus et al. 2001).
Reporter-gene assay
Reporter-gene assays were performed essentially as described previously (Bakker, Schoonus et al.
2001). In brief cells transiently co-transfected with pNF-κB-Luc (125 µg/1.10 7 cells) and pcDEF3
encoding WT or mutant H1Rs (25 µg/1.10 7 cells) were seeded in 96 well white plates (Costar) in
serum free culture medium and incubated with drugs. After 48 hrs, cells were assayed for
luminescence by aspiration of the medium and the addition of 25 µl/well luciferase assay reagent
(0.83 mM ATP. 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 µM Na2H2P2O7, 38.9 mM Tris (pH 7.8).
107 | P a g e

Chapter 6
0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100 and 2.6 µM dithiothreitol). After 30 min,
luminescence was measured for 3 sec/well in a Victor plate reader (Wallac).
Histamine H1R binding studies
Histamine H1R binding studies were performed essentially as described previously (Bakker,
Schoonus et al. 2001). In brief, transfected COS-7 cells used for radioligand binding studies were
harvested after 48hrs and homogenized in ice-cold 50 mM Na2/K-phosphate buffer (pH 7.4)
(binding buffer). The COS-7 cell homogenates were incubated for 30 min at 30 °C in binding buffer
in 200 µl with 3 nM [ 3H]mepyramine. Non-specific binding was determined in the presence of 1 µM
mianserin. The incubations were stopped by rapid dilution with 3 ml ice-cold binding buffer. The
bound radioactivity was separated by filtration through Whatman GF/C filters that had been
treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml binding buffer and
radioactivity retained on the filters was measured by liquid scintillation counting.
Analytical methods
Protein concentrations were determined according to Bradford (Bradford 1976), using BSA as a
standard. Binding and functional data were evaluated by a non-linear, least squares curve-fitting
procedure using GraphPad Prism 4 ® (GraphPad Software, Inc., San Diego, CA).
The role of I6.45 in the H1R binding pocket
108 | P a g e
Result and discussion
Using random mutagenesis methodology, we previously identified several highly Constitutively
Active Mutants (CAMs) of H1R upon mutation at position 6.40 and 6.45 in TM6 (Bakker et.al. 2008).
The preliminary studies of the H1R mutants at the position 6.45 showed the unique
pharmacological profile of the I6.45R H1R mutant. The classical radioligand for H1R,
[ 3H]mepyramine (see chemical structure at Figure 1) does not bind to the I6.45R H1R mutant.
However, the tricyclic radioligand [ 3H]doxepin (see chemical structure at Figure 1), could bind the
the I6.45R H1R mutant with an affinity of ~ 8.0 nM.
In this study, we continued to study the pharmacology of the I6.45R H1R mutant in more detail. As
shown in Table 1, the wild type H1R was well expressed in COS-7 cells (Bmax 14.8±4.6 pmol/mg
protein, n=7, p and KD 1.5±0.2 nM, n=7) as measured by radioligand [ 3H]mepyramine binding. In

The role of isoleucine at position 6.45 in the H1R
contrary to the wild type H1R, the I6.45R H1R mutant lost its binding affinity towards the
radioligand [ 3H]mepyramine in a good agreement with the previous studies [Bakker et al. 2008].
Mepyramine Doxepin Ketotifen
Figure 1 Chemical structures of H1R inverse agonists: mepyramine, doxepin and ketotifen
Table 1
Pharmacological values of the wild type H1R and I6.45 H1R mutants
Constructs
[ 3H]mepyramine [ 3H]doxepin
KD Bmax KD Bmax
nM±S.E.M. (N) pmol/mg protein
±S.E.M. (N)
nM±S.E.M. (N)
pmol/mg protein
±S.E.M. (N)
WT 1.5±0.2(7) 14.8±4.6(7) 1.5±0.2(3) 9.3±1.2(3)
I 6.45R c.n.d. c.n.d. 10.4±0.1(3) 5.1±1.1(3)
The values obtained from radioligand binding studies of the wild type H1R and I6.45R H1R mutant expressed
in COS-7 cells using either [ 3 H]mepyramine or [ 3 H]doxepin as a radiotracer. Data shown are mean values ±
S.E.M. (n, number of experiments). c.n.d. = the value cannot be determined
The I6.45 H1R mutant was, however, able to bind to the radioligand [ 3H]doxepin with Bmax and KD
values of 5.1±1.1 pmol/mg protein (n=3) and 10.4±0.1 nM (n=3), respectively. Using [ 3H]doxepin as
radiotracer, we observed that histamine binds with a high binding affinity for the I6.45R H1R
mutant (pKi 6.9±0.2, n=4), compared to the wild type H1R (pKi 5.1±0.1, n=2), whereas mepyramine
has a drastically decreased affinity for the I6.45R H1R mutant (pKi 5.2±0.2, N=3), compared to the
wild type H1R (pKi 9.2±0.1, N=2). Ketotifen (Figure 1), which is a H1R inverse agonist that contains a
tricyclic moiety similar to doxepin, binds to both the wild type H1R and the I6.45R H1R mutant in a
similar manner (Table 2 and Figure 2).
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Chapter 6
Table 2
pKi values of the wild type H1R and I6.45 H1R mutants
Constructs
110 | P a g e
pKi values
Histamine Mepyramine Doxepin Ketotifen
WT 5.1±0.1 (2) 9.2±0.1 (2) 8.7±0.3(4) 9.1±0.1 (3)
I 6.45R 6.9±0.2 (4) 5.2±0.2 (3) 8.2±0.4(5) 9.2±0.2 (4)
The values obtained from radioligand binding studies of the wild type H1R and I6.45R H1R mutant expressed
in COS-7 cells using [ 3 H]doxepin as a radiotracer. Data shown are mean values ± S.E.M. (n, number of
experiments). c.n.d. = the value cannot be determined
Figure 2 [ 3H]doxepin binding analysis of the H1R wild type or I6.45R H1R mutant
The radioligand binding assays were performed using COS-7 cells membranes transiently transfected with
the H1R wild type (A) or I6.45R H1R mutant (B) in the presence of different concentrations of histamine (•),
mepyramine (ο), doxepin (�) and ketotifen ( ). Data shown are from a representative experiments (N≥ 2)
performed in triplicate.
Although the residue I6.45 in TM6 of the H1R has previously not been implicated in the H1R ligand-
binding site, our present results suggest that it is possible that the mutation at this position may
change the structure of TM6 in such a way that it affect the binding of ligands. In addition to this,
the ligand binding studies using [ 3H]mepyramine and [ 3H]doxepin have revealed that the I6.45R
H1R mutant has low affinity for mepyramine, but still displays a high affinity for doxepin. The

The role of isoleucine at position 6.45 in the H1R
disruption of the orientation of TM6 through the I6.45R H1R mutation might disturb an important
interaction between mepyramine and F6.52, which does not affect the binding of doxepin. This
indicates that the tricyclic compounds possibly have a different binding mode compared to the
biaryl class of H1R inverse agonists such as mepyramine. This is in good agreement with previous
studies, where the binding of doxepin and related compounds was shown to be independent from
an interaction with TM 6 and showed a crucial role for only D3.32 (Nonaka, Otaki et al. 1998).
Figure 3 Basal activity of the Basal H1R activation and maximal histamine-induced NF-κB
activation
The basal activity (white bar) and maximal histamine-induced NF-κB activation (black bars) of the wild type
H1R (WT), the I6.45R and I6.45N H1R mutant and the pcDEF3 vector (MOCK) in COS-7 cells. The basal
activity of WT H1R is put at 100%. Values are mean± S.E.M. of at least 3 independent experiments.
The role of I6.45 in the H1R activation
The function of the wild type H1R was evaluated by the NF-κB reporter-gene assay as previously
reported (Bakker, Jongejan et al. 2008). The wild type H1R is constitutively active and NF-κB
activation is increased when stimulating the wild type H1R receptor with histamine (Figure 3).
Mutation of the isoleucine at position 6.45 to arginine results in a H1R mutant (I6.45R) that lost its
constitutive activity. Interestingly, despite its high affinity for histamine, the I6.45R H1R mutant
could not be activated by histamine.
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Chapter 6
Figure 4 Modulation of NF-κB and CRE activity the wild type H1R or the I6.45R H1R mutant
The reporter gene assays were performed using COS-7 cells expressing the wild type H1R or the I6.45R H1R
mutant. The transfected cells were induction by H1R ligands such as histamine (�), mepyramine ( ), doxepin (•)
and ketotifen (ο) after 48 hrs of transfection. Basal activity of each construct is put at 100%. Data shown are
from a representative experiments (N≥ 2) performed in triplicate.
Even more surprising, H1R inverse agonists that contain a tricyclic moiety such as doxepin and
ketotifen, were able to stimulate this I6.45R H1R mutant as agonists with high potencies of 7.2±0.1
(N=7) and 8.8±0.3 (N=7), respectively (Table 3 and Figure 4). Similar effects of histamine and
these tricyclic H1R ligands with the wild type H1R and I6.45R H1R mutant were also observed in a
CRE reporter gene assay (Table 3 and Figure 4). In CRE reporter gene assay, doxepin and ketotifen
were able to stimulate the I6.45R H1R mutant with potencies of 7.7±0.3 (N=3) and 8.9±0.2 (N=4),
respectively. Since reporter gene assays to trace activation of the transcription factors such as CRE
or NF-κB are very downstream of GPCR activation (Siehler 2008), we, also measured [ 3H]inositol
112 | P a g e

The role of isoleucine at position 6.45 in the H1R
phosphate formation produced by the wild type H1R and the I6.45R H1R mutant expressed in COS-7
as previously described (Bakker, Weiner et al. 2004).
Table 3
Pharmacological values of the wild type H1R and I6.45 H1R mutants
Constructs
NF-κB reporter gene assay
Histamine Mepyramine Doxepin Ketotifen
pEC50 %Effect pIC50 %Effect pIC50 %Effect pIC50 %Effect
WT 6.5±0.1(4) +435±17(4) 7.9±0.1(3) -76±3(3) 8.3±0.1(3) -53±10 (3) 8.9±0.2(4) -47±10 (4)
I 6.45 R c.n.d. c.n.d. c.n.d. c.n.d. 7.7±0.2(7)* +339±70(7) 9.0±0.1(3)* +510±118(3)
I 6.45 N 6.8±0.2(3) +470±60 (3) 7.3±0.2 (3) -79±2 (3) 8.2±0.2 (5) -62±4 (5) 9.2±0.3 (6) -66±8 (6)
Constructs
CRE reporter gene assay
Histamine Mepyramine Doxepin Ketotifen
pEC50 %Effect pIC50 %Effect pIC50 %Effect pIC50 %Effect
WT 6.1±0.1 (5) +553±96 (5) 7.2±0.1 (8) -51±6 (8) 7.9±0.3 (4) -68±4 (4) 8.7±0.1 (2) -75±4 (2)
I 6.45 R c.n.d. c.n.d. c.n.d. c.n.d. 7.7±0.3(3)* +200±55(3) 8.9±0.2(4)* +443±47(4)
I 6.45 N 6.1±0.1(4) +544±87 (4) 7.3±0.1 (9) -60±6 (9) 7.9±0.1 (2) -67±12 (2) 8.4±0.2 (3) -72±8 (3)
Constructs
Inositol triphosphate accumulation assay
Histamine Mepyramine Doxepin Ketotifen
pEC50 %Effect pIC50 %Effect pIC50 %Effect pIC50 %Effect
WT 6.6±0.2 (3) +490±85(3) 8.8±0.1 (2) -50±10(2) 9.0±0.2 (3) -45±5 (3) 8.9±0.1 (2) -42±6 (2)
I 6.45 R 5.2±0.1 (3) +56±4 (3) c.n.d. c.n.d. 8.4±0.2 (3)* +174±24 (3) 8.6±0.2 (2)* +170±10 (2)
The values obtained from reporter gene assay and [ 3H]inositol phosphate formation of the wild type H1R and
I6.45R H1R mutant expressed in COS-7 cells. Data shown are mean values ± S.E.M. (n, number of
experiments). c.n.d. = the value cannot be determined, * = pEC50
The [ 3H]inositol phosphate formation assay relies on the incorporation of [ 3H]myo-inositol into
cellular phosphatidyl-inositol 4,5-bisphosphate. Upon agonist binding to the H1R, the labeled
phosphatidyl-inositol 4,5 bisphosphate is hydrolyzed by PLC to inositol-1,4,5-triphosphate (IP3). In
the present of Li +, de-phosphorylation of IP3 to inositol is blocked, and the mass of soluble inositol
phosphates is a quantitative readout of the H1R activation. As shown in Table 3 and Figure 5,
histamine activates the wild type H1R (pEC50 6.6±0.1, N=3) and inverse agonists such as
mepyramine, doxepin and ketotifen can inhibit the basal activity of the H1R. In contrast to the wild
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Chapter 6
type H1R, the I6.45R H1R mutant showed a strongly decreased potency for histamine (pEC50
5.3±0.1, N=3). The inverse agonists with a tricyclic moiety, such as doxepin and ketotifen, showed
an agonistic effect on the I6.45R H1R mutant. These results are in a good agreement to that obtained
from the reporter gene assays.
Figure 5 [ 3H]inositol phosphate formation of the wild type H1R or the I6.45R H1R
The [ 3 H]inositol phosphate formation assays were performed in COS-7 cells expressing with the wild type
H1R or the I6.45R H1R mutated. The transfected cells were stimulated with H1R ligands such as histamine (�),
mepyramine ( ), doxepin (•) and ketotifen (ο). Basal activity of each construct is put at 100%. Data shown
are from a representative experiments (N≥ 2) performed in triplicate.
The agonistic effects of the tricyclic H1R inverse agonists on the I6.45R H1R mutant can be explained
using the β2AR-based model of the H1R (collaboration with Dr. L. Pardo). The used H1R model
explains the role of several residues that are known to be involved in the binding pocket of the H1R
such as D3.32 and W6.48 (Jongejan, Bruysters et al. 2005; Jongejan and Leurs 2005). In preliminary
molecular dynamic simulation runs, the tricyclic rings of ketotifen binds into the aromatic pocket of
the wild type H1R, formed by F5.47, F6.52, and W6.48 and thereby inhibit the conformational
transition of the W6.48 towards the active state (Figure 6) (see Chapter 1 for further reading on
the mechanism of receptor activation).
Mutation of the isoleucine 6.45 into arginine adds a positive charge in the binding pocket, thereby
inducing the polar atom of ketotifen to point towards the intracellular side and forming a hydrogen
114 | P a g e

The role of isoleucine at position 6.45 in the H1R
bond with W6.48. This network triggers the conformational transition of the W6.48 towards the
active state and leads to the activation of I6.45R H1R mutant. More experiments using homology
modeling and/or further mutation analysis need to be performed in order to clarify the role of I6.45
in the TM6 of the H1R in the receptor activation mechanisms and to explain why the tricyclic H1R
inverse agonists act as agonists at the I6.45R H1R mutant.
Figure 6 Binding of ketotifen to the wild type H1R and the I6.45R H1R mutant
The model showing the binding of ketotifen to the wild type H1R and the I6.45R H1R mutant was obtained
from Dr. L. Pardo. The H1R model was based on the crystal structure of the β2AR. The tricyclic rings of
ketotifen bind inside the aromatic pocket of the wild type H1R. The polar atom of ketotifen forms a hydrogen
bond with K5.39 and constrains the conformation of W6.48 in the inactive state. Mutation of the I6.45
creates the positive charge inside the binding pocket. The polar atom of ketotifen is forced to form a
hydrogen bond with the R6.45 residue and W6.48, thus stabilizing the active conformation of the receptor.
Besides the I6.45R H1R mutant, we also characterized other H1R mutants at this residue.
Interestingly, we have identified a high degree of constitutive activation from the mutation of
isoleucine to asparagine (I6.45N) (Figure 3). However, besides showing its high constitutive
activity, the I6.45N H1R mutant does not possess a unique pharmacological profile like the I6.45R
H1R mutant. Histamine, mepyramine, doxepin and ketotifen modulate the I6.45N H1R mutant in a
similar manner as the wild type H1R as determined by NFκB and CRE reporter gene assay (Table
2). The mechanism of constitutive activation is, however, not yet known. Possibly, the I6.45N H1R
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Chapter 6
mutant disrupts a bond between F6.44 and the TM 3 of the H1R, which was previously shown to be
involved in the activation process in rhodopsin and other biogenic amines (Donnelly, Findlay et al.
1994; Spalding, Burstein et al. 1998; Baranski, Herzmark et al. 1999; Chen, Lin et al. 2002), and
stabilizes the I6.45N H1R in its active configuration. These findings, therefore, would further
underscore the importance of the isoleucine residue at position 6.45 in the H1R activation.
116 | P a g e
Conclusion
In conclusion, we demonstrate that mutations at a single residue (I6.45) can induce multiple
receptor species such as a high constitutive active mutant (I6.45N) and, more important, an
exceptional mutant that does not respond to the endogenous ligand histamine (I6.45R) in reporter
gene assays (NF-κB and CRE) and inositol triphosphate accumulation, but could be activated by
only a particular class of synthetic H1R inverse agonists that contain tricyclic moieties such as
doxepin and ketotifen. Moreover, the I6.45R mutant shows no binding activity to mepyramine,
while it still retains high affinity for the two other tricyclic inverse agonists; doxepin and ketotifen.
This supports the previous finding (Nonaka, Otaki et al. 1998) that the biaryl H1R inverse agonists
most likely bind differently compared to the tricyclic H1R inverse agonists. These pharmacological
data, in combination with future molecular modeling studies will provide better insight on H1R
ligand binding sites and the receptor activation process.

CHAPTER
General discussion
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Chapter 7
118 | P a g e
General discussion
G protein-coupled receptors (GPCRs) consist of a single polypeptide chain of 400-500 residues that
wind seven times back and forth through the lipid bilayer connecting an extracellular amino N-
terminus to an intracellular carboxy C-terminus. They are the largest class of membrane protein in
the human genome and mediate responses from extracellular signals as diverse as biogenic amines,
lipids, peptides, proteins, odorants, pheromones, tastants, ions, and photons. GPCRs are therefore
involved in various physiological processes like smell, taste, vision, behavior, mood, regulation of
the immune system and autonomic nervous system transmission, which making them an attractive
target for the pharmaceutical industry (Jacoby, Bouhelal et al. 2006; Schlyer and Horuk 2006;
Lefkowitz 2007). As shown in Table 1, six out of top ten best selling drugs are targeted to GPCRs.
Table 1.
Examples of top selling drugs (a)
Molecular
Entity
Trade
name
Target Therapeutic
Indication
Atorvastatin Lipitor® Enzyme Hypercholesterolemia 13.5
Clopidogrel Plavix® GPCR (P2Y12) Stroke 7.3
Esomeprazole Nexium® Enzyme Dyspepsia 7.2
Salmeterol Seretide® GPCR (β2) Asthma 7.1
Etanercept Enbrel® TNFα Autoimmune diseases 5.3
Olanzapine Zyprexa® GPCR(5HT2/D1/D2) Schizophrenia 5.0
Risperidone Risperdal® GPCR (5HT2/D2) Schizophrenia 4.9
Quetiapine Seroquel® GPCR (5HT2/D1/D2) Schizophrenia 4.6
Montelukast Singulair® GPCR (LT) Asthma 4.5
Darbepoetin Aranesp® Erythropoiesis Anemia 4.4
World Sales
(US$ billions)
(a) Source: IMS MIDAS®, Dec 2007; Top ten global products 2007; All information current as of Feb 26, 2008;
The report excludes unaudited markets, and Russia, Ukraine and Belarus.
Abbreviation: P2Y12, purinergic receptor; β2, adrenergic receptor; 5HT2, hydroxytryptamine receptor; D1,D2,
dopamine receptor; LT, leukotriene receptor
Histamine H1 receptors (H1Rs) belong to the rhodopsin family of GPCRs. They are expressed
ubiquitously in smooth muscle, vascular endothelium, gastro-intestinal tract, heart, and brain. Upon
histamine binding, histamine exerts many actions related to allergic inflammation (Thurmond,
Gelfand et al. 2008) in the skin (e.g. local reddening, wheal and flare), nose (e.g. nasal congestion
and runny nose) and lower airways (e.g. bronchoconstriction). Furthermore, the H1R is also

General discussion
responsible for many of the CNS effect of histamine, such as the reduction of locomotor activity,
exploratory behavior, and cognitive functions (Inoue, Yanai et al. 1996). Knowledge regarding 3D
structural detail will facilitate the understanding of how the H1R and the other GPCRs function at
atomic level (Pierce, Premont et al. 2002; Horn, Bettler et al. 2003; Akermoun, Koglin et al. 2005;
Lundstrom 2005). However, due to the low expression in native tissues and the difficulties in GPCR
purification and crystallization (Sarramegna, Talmont et al. 2003; Lundstrom, Wagner et al. 2006),
the structural information of these important membrane proteins has been limited to crystal
structures of the naturally abundant GPCR, rhodopsin [31,54,56,71], the β-adrenergic receptors
[40-42] and the A2A adenosine receptor(Jaakola, Griffith et al. 2008). The aim of the research
described in this PhD thesis is to gain insight in structural features of the H1R using techniques such
as mass spectrometry and site-directed mutagenesis in combination with homology modeling
studies. Using a new in vitro cell free expression systems, we were able to express milligrams of the
H1R protein within 8-12 hours. The protein obtained can be purified by a simple and cost effective
IMAC chromatography and reconstituted into artificial membranes prepared from asolectin lipids.
The purified H1R was analyzed by ligand binding analysis using [ 3H]mepyramine as a radiotracer.
The cell free-produced H1R protein retains its binding activity towards the inverse agonist
mepyramine and its endogenous ligand histamine and does not bind to H2, H3 and H4 ligands
(Chapter 3). The rapid generation of sufficient amount of the H1R protein using this in vitro cell free
expression system allowed us to use the H1R protein in mass spectrometry (MS) analysis. With its
high sensitivity, together with continuous improvements in technology and software, MS
technology has become an important tool in the sequencing and characterizing of proteins and
protein complex. By employing an acid labile surfactant during the tryptic digestion prior to
MALDI-ToF MS analysis, we were able to obtain high sequence coverage of the H1R, including five
out of seven transmembrane (TM) domains (Chapter 3). Following the same approach for protein
production using an in vitro cell free expression system using MALDI-ToF MS, we were able to
produce milligrams amount of the H4R, a new potential target for drug discovery. Coverage of
~50% amino acid sequence of the H4R, including three out of seven TM domains, was obtained
(Chapter 3). Future works should focus on the improvement of the percent coverage of the amino
acid sequence and the functional characterization of the purified and reconstituted H4R protein.
Employing a similar mass spectrometric analysis approach, we were also able to obtain the high
sequence coverage of the H1R protein expressed from baculovirus/insect cells expression system as
previously described by Ratnala and co-workers [126]. Using MS analysis, we show here for the
first time that an asparagine residue in the H1R protein is subjected to the N-linked glycosylation
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Chapter 7
(Chapter 4). These data suggests that MS technology is a promising tool for the characterization of
higher order protein structures, post-translation modifications, and receptor-ligand binding
interactions.
In Chapter 4, we also report on a preliminary MS experiment in map residues that involved in the
H1R-ligand binding. The ligand phenoxybenzamine was found to irreversibly bind to the H1R,
expressed by baculovirus/insect cells expression system and the cysteine residue (Cys302) located
in the intracellular loop of the H1R was identified by MS analysis to be alkylated by
phenoxybenzamine. However, based on the structured model of the rhodopsin family of GPCRs,
including the H1R, the binding pocket is thought to reside within the TM domains, such as TM3, TM5
and TM6 [See Chapter 1 for further reading]. Furthermore, it has also been shown that the possible
cysteine reside that is involved in the binding of phenoxybenzamine is located at position 3.36 of
TM3 in the α-adrenergic receptor [256]. Therefore, we speculate that our preliminary finding that
we obtained from mapping of phenoxybenzamine and the H1R (at the Cys302) is the result from the
non-specific reaction of phenoxybenzamine [314]. Recently, the works of Espinoza-Fonseca [312]
and Nicholls [313] have, however, suggested possible binding sites of GPCR ligands to the
intracellular region. It is therefore interesting to further investigate the binding site of
phenoxybenzamine and the H1R. For future MS analysis, the protocol regarding the
phenoxybenzamine treatment and the H1R sample preparation can be adjusted to minimize a
potential non-specific reaction. It is also noteworthy to study the intracellular region of the H1R as a
possible binding site by mutation analysis together with homology modeling studies.
In Chapter 5, we have combined the latest structural insights in GPCR structures, computation
modeling and site-directed mutagenesis of the H1R to study the role of the TAS(I/V) motif at
positions 3.37-3.40 in TM3 in the process of receptor activation. We have designed point mutations
that modulate the strength of intra-molecular interactions and the side chain rotamer distribution
of specific residues, and measured their effects on constitutive agonist-independent H1R activity.
We have shown here that the threonine T3.37 interacts with TM5 in the inactive state of the H1R,
while the serine S3.39 changes its conformation upon receptor activation from the inactive g+ to
the active g- or t conformer, together with S3.36, to achieve new water-mediated inter-helical
interactions with TM2 and TM7 that stabilize the active state. We also provided the first evidence
that the hydrophobic side chain at position 3.40, which is highly conserved in the rhodopsin family
of GPCRs, plays a key role in facilitating the histamine-induced relocation of TM5. Mutation of I3.40
120 | P a g e

General discussion
to either Ala or Gly abolished the constitutive activity of the H1R and well as the histamine-induced
receptor activation. A thorough understanding on how a ligand induced the receptor will have a big
impact on our understanding of the molecular aspects of the H1R and the related family
A GPCRs. In 2008, Bakker and co-workers [62] have employed random mutagenesis studies and
identified three residues in TM6 (I6.40, A6.43 and I6.45) as sites prone to yield high constitutively
active H1R mutants. Following their work, we have found the I6.45R H1R mutant to possess a
unique pharmacological profile (Chapter 6). The I6.45R H1R mutant loses its binding activity
towards the classical biaryl H1R inverse agonist mepyramine, but still retains its affinity towards
tricyclic H1R inverse agonists such as doxepin and ketotifen, supporting the previous finding by
Nonaka and co-workers[253] that the H1R inverse agonists possibly bind differently to the H1R. The
I6.45R H1R mutant also loses its basal activity and cannot be further activated by histamine as
measured by reporter gene and [ 3H]inositol formation assays, despite its high affinity towards
histamine. Surprisingly, the mutant can only be activated by doxepin and ketotifen, the tricyclic H1R
inverse agonists. Here, we propose a possible hypothesis why the I6.45R H1R mutant is activated by
ketotifen using a H1R model based on the crystal structure of the β2 adrenergic receptor. The
arginine R6.45 in the H1R mutant provides a positive charge in the binding pocket, thereby inducing
the polar atom of tricyclic inverse agonists such as ketotifen to point towards the intracellular side
and to trigger the conformational transition of the W6.48 towards the active state via the hydrogen
bond network.
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Summary
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124 | P a g e
Summary
Histamine is a biogenic amine well-known for its role in allergic reactions. When histamine is
released in the body, for example, after specific antigens, such as pollen or dust, come in contact
with mast cells, it exerts its effect by binding to histamine receptors. Histamine receptors are G
protein-coupled receptors (GPCRs) or seven-transmembrane domain (7TM) receptors, which
represent the largest class of membrane-bound proteins in the human genome. Some of the best-
selling drugs on the market today such as Allegra® and Claritin® target histamine receptors. So far
four subtypes of histamine receptors have been discovered and cloned, and the research described
in this thesis is mainly focused on the histamine H1 receptor (H1R). Even before the cloning of the
human H1R, scientists have been able to develop antagonists that potently block histamine from
binding to the H1R. Through the years a steady increase in the structural knowledge of the
receptor, has led to increases in selectivity and strength of these antagonists, while at the same time
led to a reduction in side effects such as sedation. Pharmaceutical companies rely heavily on
structure-based design for lead optimization in drug discovery, especially if X-ray crystal structures
of a target protein are available. It is widely used in drug discovery for soluble proteins, because
they are more straightforward to isolate, purify and crystallize in comparisons to membrane
proteins. Despite the importance of membrane proteins such as GPCRs as drug targets, their
structure determination has been very limited compared to soluble proteins. The low success rate
is due to the difficulties in obtaining large amounts of high quality proteins. The research described
in this thesis was aimed to provide a better understanding of GPCR structure in general and of the
human H1R in specific, through large scale production of the H1R and subsequent purification and
structural analysis by mass spectrometry. Chapter 1 gives a brief overview of the current
knowledge of GPCRs and H1R in particular. Chapter 2 provides a systematic and thorough review of
the different options available to express and purify GPCRs, including the H1R protein. In Chapter 3,
we described how we have made use of a well established baculovirus/insect cell expression
system, and an in vitro cell free expression system to produce large amounts of the human H1R and
H4R protein. Moreover, this chapter also shows details in the use of immobilized metal affinity
chromatography (IMAC) to purify the receptors after production, as well as our endeavors to obtain
the highest possible sequence coverage of the two receptors by MALDI-ToF and LC-MS/MS. Having
established a successful and reproducible method to obtain large and pure enough quantities of the
human H1R protein allowed us to continue our goal of improving the structural knowledge and
binding pocket of the H1R. In chapter 4, we describe how we employed MS analysis to discover that
an asparagine residue in the N-terminus of the H1R (Asn5) is glycosylated in insect cells, which had

Summary
been used in conjunction with the baculovirus/insect cell expression system. Moreover we were
able to locate the residue to which phenoxybenzamine, an irreversible H1R antagonist, covalently
binds to the H1R by using MS analysis, thereby showing the great potential of our methods in the
production, purification and MS analysis of the H1R protein could have on structural analysis of
GPCRs. It is known that GPCRs can become activated in the absence of ligands. The degree of this
constitutive activity can also be modulated by single point mutations of the receptor. Constitutive
activity of receptors can be used to investigate which residues in a GPCR contributes to receptor
activation and how. In chapter 5 we used site directed mutagensis of the H1R to identify residues
that are involved in activation of the receptor. Our results suggest that T3.37 interacts with TM5 in
the inactive state of the receptor, while S3.39 changes conformation upon receptor activation to
achieve, together with S3.36, new water-mediated inter-helical interactions with TM2 and TM7 that
stabilize the active state. In addition, we provide the first evidence that the hydrophobic side chain
at position 3.40, highly conserved in the rhodopsin family of GPCRs, plays a key role in facilitating
the agonist-induced relocation of TM5. Mutation of I3.40 to either alanine or glycine, i.e. removing
the bulky side chain at this position, abolishes the constitutive activity of the H1R, the effect of
constitutive-activity increasing mutations, as well as the histamine-induced receptor activation. In
chapter 6 we describe our detailed investigation of a point mutated version of the H1R. Previous
research had identified an isoleucine residue at position 6.45 of the H1R to be prone to yield high
constitutively active mutants. We observed that the I6.45R mutant exhibits an exceptional
pharmacological profile as it no longer gives a functional response when stimulated with the
endogenous ligand histamine, but only responds to a particular class of synthetic H1R inverse
agonists that contain tricyclic moieties such as doxepin and ketotifen. Mepyramine, an inverse
agonist belonging to the class of biaryl H1R inverse agonists, is also unable to bind to the I6.45R
mutant, suggesting that these two classes of H1R inverse agonists may bind the H1R in a different
manner.
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Samenvatting
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128 | P a g e
Samenvatting
Histamine is een amine, dat door het lichaam wordt geproduceerd. Het is voornamelijk bekend voor
zijn grote rol in allergische reacties. Wanneer histamine vrijkomt, bijvoorbeeld nadat een antigen,
zoals pollen, in contact komt met een mastcel, kan de amine binden aan histaminereceptoren en zo
een effect in de cel bewerkstelligen. Histaminereceptoren zijn G-eiwit gekoppelde receptoren
(GPCRs) of zeven transmembraan domein (7TM) receptoren. Deze receptoren vormen de grootste
klasse van celmembraangebonden eiwitten. Verscheidene “blockbuster” medicijnen richten zich op
deze receptoren, inclusief een paar die zich specifiek op de histamine H1 receptor (H1R) richten
(Allegra, Claritin). Tot dusver zijn er vier verschillende histaminereceptoren ontdekt en gekloond.
Het onderzoek dat voor dit proefschrift is uitgevoerd is echter voornamelijk gericht op de H1R . Al
lang voordat de H1R gekloond was, was het wetenschappers gelukt om antagonisten, die de binding
van histamine aan de H1R kunnen voorkomen, te creëren. Over de jaren is de selectiviteit en
affiniteit van deze antagonisten toegenomen en tegelijkertijd zijn vervelende bijwerkingen als
slaperigheid afgenomen door een immer groeiend inzicht in de structuur van de H1R.
Pharmaceutische bedrijven zijn erg afhankelijk van de kennis van de structuur van de receptor voor
de ontwikkeling en verbetering van geneesmiddelen. Het verkrijgen van een X-ray kristalstructuur
is erg informatief voor dit doeleinde, en dit wordt dan ook veel toegepast, met name voor eiwitten
die gemakkelijk in oplossing blijven. Deze eiwitten zijn in tegenstelling tot GPCRs makkelijker te
produceren en op te zuiveren en uiteindelijk te kristalliseren. Ondanks de grote interesse in GPCRs
voor de ontwikkeling van geneesmiddelen is het vooralsnog moeilijk om deze receptor-eiwitten te
kristalliseren of überhaupt goede structuur informatie te vergaren. Een van de redenen hiervoor is
dat het lastig is om deze receptoren in grote hoeveelheden te produceren. Het onderzoek dat wordt
beschreven in dit proefschrift was gericht op het verkrijgen van een beter inzicht de structuur van
GPCRs en specifiek de structuur van de H1R, door middel van het produceren van de H1R op grote
schaal en het vervolgens analyseren van de gezuiverde receptor via massaspectrometrie. Hoofdstuk
1 bevat een kort overzicht van de huidige kennis op het gebied van GPCRs. Hoofdstuk 2 geeft een
uitgebreid en systematisch overzicht van alle opties en methoden die beschikbaar zijn voor het
produceren en zuiveren van grote hoeveelheden GPCRs. In dit proefschrift hebben we verschillende
methoden gebruikt om grote hoeveelheden H1R te verkrijgen. We hebben gebruik gemaakt van het
baculovirus systeem, wat een veel gebruikte methode is voor dit doeleinde. We hebben echter ook
een relatief nieuwe methode gebruikt. Deze methode is gebaseerd op een cell-vrij
expressiesysteem. In hoofdstuk 3 beschrijven we hoe we het baculovirus en het cell-vrije
expressiesysteem hebben gebruikt om H1R en H4R te produceren. Verder beschrijven we hoe we de

Samenvatting
receptors hebben gezuiverd via een geimmobiliseerde metaal-affiniteitschromotografie en onze
pogingen om de hoogste dekking te verkrijgen in de structuuranalyse van de receptoren met
behulp van MALDI-TOF en LC MS/MS.
Nadat het ons gelukt was om een succesvolle en reproduceerbare methode op te zetten voor de
productie van H1Rs, konden we ons richten op ons doel een beter inzicht in de structuur van de H1R
te verkrijgen. In hoofdstuk 4 beschrijven we hoe we massaspectrometrie hebben gebruikt om te
ontdekken dat een asparagine in de amino-terminale staart van de H1R (Asn5) is geglycolyseerd in
insectencellen. Het was ook mogelijk om een aminozuur, dat betrokken is bij de onomkeerbare
binding van de H1R antagonist phenoxybenzamine aan de receptor te lokaliseren. Hiermee konden
we ook het grote potentieel van onze methode aantonen, voor het analyseren van de structuur van
de bindingsplaats van geneesmiddelen aan de H1R en mogelijk ook andere GPCRs.
Het is bekend dat GPCRs ook zonder de binding van een agonist in een geactiveerde staat kan
bestaan. De sterkte van deze constitutieve activiteit kan gemanipuleerd worden met behulp van
puntmutaties in de receptor. Constitutieve activiteit van receptoren kan worden gebruikt om de
aminozuren, die een rol spelen bij de activering van de receptor, te bestuderen. Voor hoofdstuk 5
hebben we gebruik gemaakt van zogenaamde ‘site-directed’ mutagenese om aminozuren in de H1R
die betrokken zijn bij de activatie van de receptor te identificeren. Onze resultaten suggereren dat
de T3.37 een interactie aangaat met TM5 in de inactieve staat van de receptor. Wanneer de
receptor geactiveerd wordt, verandert de conformatie van S3.39, zodat dit residue samen met S3.36
een water-geassisteerde interactie aangaat tussen TM3, TM2 en TM7. Deze interactie stabiliseert de
actieve staat van de receptor. Tegelijkertijd leveren we ook het eerste bewijs dat de hydrofobische
zijketting op positie 3.40 een grote rol speelt in het faciliteren van de herschikking van TM5 nadat
de receptor is geactiveerd. Door het verwijderen van de grote zijketting rond 3.40 door mutaties
van I3.40 naar een alanine of een glycine, wordt zowel de constitutieve- als ook de agonist-
geïnduceerde acitiveit van de H1R tenietgedaan.
In hoofdstuk 6 beschrijven we ons onderzoek van een specifieke puntmutatie in de H1R. Eerder
onderzoek had een isoleucine op positie 6.45 geïdentificeerd, die erg vatbaar was om mutanten te
creëren met hoge constitutieve activiteit. We observeerden dat de I6.45R-mutant een opmerkelijk
farmacologisch profiel had. De mutant reageerde niet meer op de binding van histamine, maar
enkel op binding van een specifieke klasse van synthetische H1R inverse-agonisten met een
tricyclisch karakter, zoals doxepine en ketotifen. Mepyramine, een andere klasse H1R inverse-
129 | P a g e

Samenvatting
agonist, kon echter niet aan deze mutant binden. Dit doet vermoeden dat deze twee klassen van
inverse agonisten op een verschillende manier aan de H1R binden.
130 | P a g e

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Acknowledgements
Jib
[Type the company name]
[Pick the date]

Because there is no “I” in “TEAM”…
My Ph.D. years at the Vrije Universiteit and this
thesis have had mentorship from numerous
individuals both from within the university and
outside of it. It is to these individuals that my
heartfelt gratitude and thanks go out to, for
without their help, this thesis would not have
seen its ISBN number.
The supervising team
My promoter, Prof. Dr. Rob Leurs, thank you for
giving me a chance to do my Ph.D. study in your group.
Your knowledge and logical way of thinking have been
of great value for me and have provided a good
structure for this thesis. I also would like to express
my gratitude to my co-promoter, Dr. Remko Bakker,
thank you for your guidance and your promptly
responses to my questions, drafts and email through
these years. You are the one who put the paper from
Ishihara (the first paper showing that GPCRs can be
expressed by cell free expression system) in my
mailbox and 3 years later, we finally made it happen
with our H1R protein. I am still keeping that bottle of
champagne you gave me as the trophy of our victory.
This work would not have been possible without Prof.
Dr. Wim de Grip, Dear Wim, your expertise and
connections in membrane protein biochemistry and
biophysics are very valuable and I am so fortunate to
be supervised by you. My thanks also go to the
members at the NCMLS, Nijmegen. Arthur, thank you
for helping me with FT-IR. Jenny, thank you for
teaching me all the techniques related to BVES. And
very special thanks to Petra, Dear Petra; besides
showing me how to purify membrane proteins, you
are also my guardian angel. Thank you for making me
feels like home in Nijmegen. I deeply enjoyed your
company and a free-pass to the pit in Assen.
I would like to thank Dr. Paul Hensbergen and his
team Crina and Anne for their contribution in chapter
3 and 4 in this thesis. Dear Paul, you are my MS guru. I
really enjoyed my visits to Leiden and our telephone
conversations. As much as you can be very serious,
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you can also laugh very easily. It really helps making
such a good vibe in our collaboration.
I am also indebted to Prof. Dr. Leonardo Pardo and
his team Dr. Xavier Deupi and Ivan Torrecillas for
their contribution in chapter 5 and 6 in this thesis.
Without their help in structure modeling, I probably
would not be able to solve the puzzles regarding our
H1R mutants.
I wish to express my thank to Prof. Dr. Raymond
Booth, whose valuable comments and funding has
been of great support to our work. My thanks also go
to the reading committee whose name has not been
mentioned yet. Thank you Prof. Dr. Martine Smit, Dr.
Astrid Alewijnse and Dr. Marco Siderius for
reviewing my thesis. Also many thanks to my former
colleagues, Dr. Martijn Bruyster and Dr. Aldo
Jongejan for their valuable comments and advice
regarding my work with the H1R.
The scientific supporters
Richard van Rijn, my dear friend, your multi-tasking
and self-discipline has never failed to impress and
inspire me. Thank you for having faith in me, for
always encouraging and keeping me focused, for
spending hours cheering me up through the phone
lines, for being there through my good and bad times,
for letting me be your friend, for staying friend with
me and for being the best, best friend in the world.
Gerold Bongers, thank you for always taking me
along on your motorbike. For you it is adrenaline
kicks, for me it is meditations (since I manage to fall
asleep anyway). I also appreciate that you show up at
every running races. I probable never said it before
but you are the best supporters and definitely
deserved some credits for those medals.
Laura de Wit, You always greet me with a warm smile
and open arms every time I dropped by your office.
Thank you for your friendship and kind helps in many
different ways.
Andre van Marle and Tonio (JOSÉ ANTONIO ARIAS
MONTAÑO), thank you for all your helps and

comments, especially in my first year. Dear Andre, the
book “Cell Free expression system” that you gave me
will always be my holy bible. Dear Tonio, what you
taught me on my first week is a good basis of my
purification work up till now. Also, thank you for the
lovely present from Mexico. (Although, it is funny to
see that other people in the lab got bottles of tequila,
but I got a Barbie doll.)
Herman Lim and Atilla Akdemir, thank you for your
friendship and patience when listening to all my
miserable stories. Dear Herman, I enjoyed the time we
shared the office together, ate all those exotic food,
and spaced out on the cola breaks. Let’s make our
friendship a decade! Dear Atilla, my VU gym partner,
you know that I was so strict on you during the yoga
class or at the gym, because of “good intention and
tough love”, right? And now stop calling me B****!!!
Dennis Verzijl and Henry Vischer, thank you for
your helps and comments through these years. Dear
Dennis, special thanks to the birthday present you
gave me last year. It was either “Life-threatening” or
“Life-changing” experience. Fortunately, it is the latter
one in my case. I cannot wait for more goodies. Dear
Henry, aka Nemo, I love it when you sometimes
dropped by my room (N-249) for a quick chat and also
many thanks to your comments when I had to prepare
my presentations. I hope your next holiday destination
will be Thailand again. Besides the temple full of
tigers, we also have monkeys and other exotic animals,
you know!
Saskia Hulscher and Obbe Zuiderveld, thank you for
your numerous helps. Dear Saskia, besides helping me
with all the ordering of expensive kits that I needed,
you also provided me something with much greater
value in all these years; “a friendly smile, a hello every
time we met and the warm friendship”. Dear Obbe,
thank you for trying to treat me very gently, although I
sometimes make a mess in the RNC. As a sign of my
appreciation, I will make sure to serve good beverages
at my reception.
My special warm gratitude also go to my former
bench-mates and now FB friends; David Maussang,
Leontien Bosch and Erik Slinger. I really enjoyed
your presence and company. You made coming to the
149 | P a g e
lab much more fun and time did go by quickly. Dear
David, since this is possibly the only book I am going
to write, I will come out of my closet now; you are my
number one “lieveling” in the lab. Dear Leo and Erik,
thank you for your supports and positive energy. I
know I can always count on you, especially when it
comes to moving. Thank you!
My “Die-hard” yoga team; Anne, Azra, Chimed, Kim,
Ellen, Meritxel, Marija, Petra, Saskia (N) and
Sabrina. Thank all of you for the lovely and vibrant
yoga evenings we shared together at the VU library,
the coffee room and my apartment. Chest up! Stomach
in! butt tights! Straight! and strong! Now another 10
sun salutations! No need to say more, girls. Your
enthusiasms really made it happened!
My students; Tofan, Laura and Kim, I hope you did
learn something about protein purification…besides
scraping million of dishes. Thank you for all the fun,
good laughs and youthful energy you bring along,
especially Kim. Lieve Kim, thank you for being my
personal assistant, my secretary, my dear friend, my
sister and most importantly thank you for teaching me
that I would never be the mature one in our
friendship, but that it was also fine because you totally
had it covered!
I also wish to express my warm regards to all the other
colleagues and the interns that I have encountered
through these years; Regina (thank you for the
friendship, I hope to visit you one day in Ghana), Jan
Paul Bebelman (thanks for all the helps and many
nice conversations we have), Jane van Heteren (I did
my first 7 hills running because of you), Fiona
Shenton (although only 2-3 days, but you taught me
so many tips for the running), Mirabella (my all-time
beautiful girl), Martine (MvK, thanks for playing
tennis with me), the twin sisters (Angelique and
Payman) and the twin brothers (Lars and Deni) who
always put smiles on my face and many more of you
that I cannot mention all in this chapter. Thank you!
The “thuisfront”
Dear Anneli Lundgren, you are one of the most
kindhearted people I know-genuinely. Even though,

we are far apart for many years already, somehow I
feel as if the distance just enriches our friendship.
Thank you for all the postcards you sent to me from all
over the world. I can hardly wait to see your next
adventure. Dear Maria Edman, thank you for showing
me how to be best friend to somebody. Dear Yanchao
Huang and Mika, thank you for always trying to keep
in touch. I hope one day our path will be crossed again.
Dear Molly Ackerman, thank you for being a good
role model for me, you are my artist, my inspiration
and my special friend that I always look up to. My girls
Siew Hoon Chan, Lianne Begga, and Julia
Schorestene, you probably do not know each other,
but you all shared very special characters that I
appreciate so much in a friend. Thank you for always
be there for me when I am down and for showing me
how cool it is to be independent!
Many thanks also go to my Thai contacts:
entire extended family members for providing a loving
home for me. Without their love, supports, and
ขอบคุณพี่หน�อย พี่ต�าย พี่เอ�ม อาจารย�ตั้ม encouragements อาจารย�แดง it would have been impossible for me
เจ�าแม็คน�องสุดที่รักของพี่และครอบครัวของแม็คพี่เล็ก
to get through the decade of cold and lonely time in
และพี่ๆที่ร�านสบายๆและร�านศรีโสธรนะคะ Europe.
ขอบคุณสําหรับอาหารอร�อยๆและไออุ�นที่มีให�จิ๊บเสมอมา
พี่โจพี่โบเปรียบเสมือนคุณพ�อคุณแม�คนที่สองขอ
ขอบคุณที่รักและดูแลหนูตลอดมา
Special thanks to two of my paranimfen; Sonia Faut,
my sweetheart, I wish to thank you for the very
intense six months we had together in Basel. I
genuinely feel as though I have found a soul sister in
you. Thank you for being my friend, for showing me
that it takes 8 doors between 1978 and 1985, for
always checking if I am working on the thesis or not,
for making the original French pancake for me, for
learning me how to pronounce Mulhouse correctly, for
suffering in the gym and firmenlauf with me, for
hugging me when I cry and for making time for my
promotion. “Sonia, ma chérie, Tu me fais sourire. Je
t'aime de tout mon coeur.” Alejandro Bernadis, Dear
Alejandro, you are probably the happiest man on earth
and I always have a deep respect on you. I feel warm,
comfortable and calm around your presence (which is
probably what I need the most before the defense). It
is my honor that you agreed to wear the penguin suit
and be my paranimf. Muchísimas gracias!
A very special thank is due to the Bikram yoga school
of Amsterdam. It started out as “Jib’s new challenge”,
but the integrity and the devotion of the fellow yogis
150 | P a g e
towards the practice deeply inspired me and
completely changed my attitude and lifestyles. Also
from there, I got a chance to get to know many people
outside the scientific world and even more
importantly some of them have become the dearest of
my heart.
Dear Rodrigo, thank you for beating my ego down to
nothing, so I could rise up completely as the true
person. Thank you for taking risks with me, for
embracing our differences, for continually surprising
me with how wonderful your ideas are, for making me
realize that having those huge dreams is more than
okay, for giving your everything to everything and
most of all, thank you for being “you”.
I wish to thank my sister พี่โจ
พี่โจ , my brother พี่โบ พี่โบ
พี่โบ and my
หนูขอให�เรามีกันและกันตลอดไป
Lastly and most importantly, I wish to express my
deepest gratitude to my parents, Mongkhol and
Nonthawan Sansuk. You brought me into this
world, raised me with all your love and care,
supported me to your utmost ability, taught me to
be a good person, and loved me unconditionally.
For you, a thousand times over.
To you, I dedicate this thesis.
คุณพ�อคุณแม�คะ
หนูทําสําเร็จแล�วนะคะความฝ�นของเรา
ต�อไปนี้เราจะไม�ต�องอยู�ไกลกันแล�วนะคะ
หนูรักคุณพ�อคุณแม�มากที่สุดในโลก

“The foot feels the foot when it feels the ground.”
Lord Buddha