THE INFANCY OF SIGNAL TRANSDUCTION—GTP STIMULATION ...

Classic Experiment
13.1
THE INFANCY OF SIGNAL
TRANSDUCTION—GTP STIMULATION
OF CAMP SYNTHESIS
In the late 1960s the study of hormone action blossomed following the discovery
that cyclic adenosine monophosphate (cAMP) functioned as a second messanger,
coupling the hormone-mediated activation of a receptor to a cellular response. In
setting up an experimental system to investigate the hormone induced synthesis of
cAMP, Martin Rodbell discovered an important new player in intracellular signalling
— guanosine triphosphate (GTP)
Background
The discovery of GTP’s role in regulating signal transduction
began with studies on how glucagon and other hormones
send a signal across the plasma membrane that
eventually evokes a cellular response. At the outset of
Rodbell’s studies, it was known binding of glucagon to
specific receptor proteins embedded in the membrane
stimulates production of cAMP. The formation of cAMP
from ATP is catalyzed by a membrane bound enzyme
called adenyl cyclase. It had been proposed that the action
of glucagon, and other cAMP stimulating hormones,
relied on additional molecular components that couple
receptor activation to the production of cAMP. However,
in studies with isolated fat cell membranes known as
“ghosts,” Rodbell and his coworkers were unable to provide
any further insight into how glucagon binding leads
to an increase in production of cAMP. Rodbell then began
a series of studies with a newly developed cell-free system,
purified rat liver membranes, which retained both membrane-bound
and membrane-associated proteins. These
experiments eventually led to the finding that GTP is
required for the glucagon-induced stimulation of adenyl
cyclase.
The Experiment
One of Rodbell’s first goals was to characterize the binding
of glucagon to the glucagon receptor in the cell-free rat
liver membrane system. First, purified rat liver membranes
were incubated with glucagon labeled with the radioactive
isotope of iodine ( 125 I). Membranes were then separated
from the unbound [ 125 I]glucagon by centrifugation. Once
it was established that labeled glucagon would indeed
bind to the purified rat liver cell membranes, the study
went on to determine if this binding led directly to activation
of adenyl cyclase and production of cAMP in the
purified rat liver cell membranes.
The production of cAMP in the cell-free system
required the addition of ATP, the substrate for adenyl
cyclase, Mg 2 , and an ATP-regenerating system consisting
of creatine kinase and phosphocreatine. Surprisingly,
when he glucagon binding experiment was repeated in the
presence of these additional factors, Rodbell observed a
50 percent decrease in glucagon binding. Full binding
could be restored only when ATP was omitted from the
reaction. This observation inspired an investigation of the
effect of nucleoside triphosphates on the binding of
glucagon to its receptor. It was shown that relatively high

(i.e., millimolar) concentrations of not only ATP but also
uridine triphosphate (UTP) and cytidine triphosphate
(CTP) reduced the binding of labeled glucagon. In contrast,
the reduction of glucagon binding in the presence of
GTP occurred at far lower (micromolar) concentrations.
Moreover, low concentrations of GTP were found to stimulate
the dissociation of bound glucagon from the receptor.
Taken together, these studies suggested that GTP alters
the glucagon receptor in a manner that lowers its affinity
for glucagon. This decreased affinity both affects the ability
of glucagon to bind to the receptor, and encourages the
dissociation of bound glucagon.
The observation that GTP was involved in the action
of glucagon led to a second key question: Can GTP also
exert an affect on adenyl cyclase? Addressing this question
experimentally required the addition of both ATP, as a
substrate for adenyl cyclase, and GTP, as the factor being
examined, to the purified rat liver membranes. However,
the previous study had shown that the concentration of
ATP required as a substrate for adenyl cyclase could affect
glucagon binding. Might it also stimulate adenyl cyclase?
The concentration of ATP used in the experiment could
not be reduced, because ATP was readily hydrolyzed by
ATPases present in the rat liver membrane. To get around
pmols cAMP
1000
Glucagon + GTP
800
600
400
Glucagon
200
Basal
0 0
5 10 15 20
Minutes
Effect of GTP on glucagon-stimulated cAMP production from
AMP-PNP by purified rat liver membranes. In the absence of GTP,
glucagon stimulates cAMP formation about twofold over the basal
level in the absence of added hormone. When GTP also is added,
cAMP production increases another fivefold. [Adapted from M.
Rodbell et al., 1971, J. Biol. Chem. 246:1877.]
this dilemma, Rodbell replaced ATP with an AMP analog,
5¿
-adenyl-imidodiphosphate (AMP-PNP), that can be converted
to cAMP by adenyl cyclase, yet is resistant to
hydrolysis by membrane ATPases. The critical experiment
now could be performed. Purified rat liver membranes
were treated with glucagon both in the presence and
absence of GTP, and the production of cAMP from AMP-
PNP was measured. The addition of GTP clearly stimulated
the production of cAMP when compared to
glucagon alone (see Figure) indicating that GTP promotes
not only the binding of glucagon to its receptor but also
the activation of adenyl cyclase.
Discussion
Two key factors led Rodbell and his colleagues to detect
the role of GTP in signal transduction, whereas previous
studies had failed to do so. First by switching from fat
cell ghosts to the rat liver membrane system, the Rodbell
researchers avoided contamination of their cell-free system
with GTP, a problem associated with the procedure
for isolating ghosts. Such contamination would mask the
effects of GTP on glucagon binding and actviation of
adenyl cyclase. Second, when ATP was first shown to
influence glucagon binding, Rodbell did not simply accept
the plausible explanation that ATP, the substrate for
adenyl cyclase, also affects binding of glucagon. Instead,
he chose to test the effects on binding of the other common
nucleoside triphosphates. Rodbell later noted that he
knew commercial preparations of ATP often are contaminated
with low concentrations of other nucleoside triphosphates.
The possibility of contamination suggested to him
that small concentrations of GTP might exert large
effects on glucagon binding and the stimulation of adenyl
cyclase.
This critical series of experiments stimulated a large
number of studies on the role of GTP in hormone action,
eventually leading to the discovery of G proteins, the
GTP-binding proteins that couple certain receptors to the
adenyl cyclase. Subsequently, an enormous family of
receptors that require G proteins to transduce their signals
were identified in eukaryotes from yeast to man. These G
protein–coupled receptors are involved in action of many
hormones as well as in a number of other biological activities
including neurotransmission and the immune
response. It is now known that binding of ligands to their
cognate G protein–coupled receptors stimulates the associated
G proteins to bind GTP. This binding causes transduction
of a signal that stimulates adenyl cyclase to produce
cAMP and also desensitization of the receptor, which
then releases its ligand. Both of these affects were
observed in Rodbell’s experiments on glucagon action. For
these seminal observations, Rodbell was awarded the
Nobel Prize for Physiology and Medicine in 1994.