New Modes of GPCR Signalling

ABSTRACT
mGlu Receptors : Complex Allosteric Machines to Tune Up Synaptic
Transmission
Philippe Rondard1, Etienne Doumazane1, Pauline Scholler1, Eric Trinquet2, Sébastien
Granier1 & Jean-Philippe Pin1
1 CNRS UMR5203, INSERM U661, University of Montpellier, Department of Molecular
Pharmacology, Institute of Functional Genomics, Montpellier, France.
2 CisBio International, Bagnols/Cèze , France
The G-protein coupled receptors activated by the neurotransmitter glutamate (mGluRs)
are made up of two subunits covalently linked by a disulfide bridge. Each protomer
comprises an extracellular domain that binds agonists and a transmembrane heptahelical
domain responsible for G-protein activation. The general organization of mGluRs at the
cell surface, whether they are limited to dimers, or organized into high-order oligomers,
as well as the functioning of the dimeric receptors remain unclear.
Here, we first examined whether mGluRs can assemble into heteromeric complexes. To
that aim we describe a new approach enabling the specific labeling of two cell surface
proteins carrying SNAP- or CLIP-tags, with two distinct fluorophores compatible with
time-resolved FRET. This approach allowed us to quantify both homomeric and
heteromeric populations. Our data revealed that some, but not all pairs of mGluRs can
indeed form heteromeric entities, and this is further supported by biochemical analysis
and functional complementation studies. By FRET competition and saturation analysis,
we show that these complexes are limited to heterodimers. In addition to describing a
new way to analyze cell surface receptor complexes, our data reveal a new possible
level of complexity within the mGluR family.
Second, whereas dimerization of the extracellular domains is essential for the activation,
it is not known if the function of the transmembrane domains also depends on
dimerization. To address this question, we analyzed the G protein coupling of purified
mGluR membrane domain reconstituted into phospholipid bilayer nanodisc with a
controlled stoichiometry: one or two protomers per disc. Nanodiscs containing only one
mGluR membrane domain are able to activate G proteins in response to a positive
allosteric modulator, demonstrating that monomeric mGluR membrane domain can fold
and function independently from the rest of the receptor. These results suggest that
dimer requirement in mGluR function originate from an obligate communication
between the extracellular and transmembrane domains in the receptor for activation by
glutamate, rather than a necessity for specific association between the two membrane
domains.