My role began the day Martin Rodbell took me from his home to his laboratory in Building 10 of the NIH Campus in Bethesda. I had arrived the evening before from Argentina with a brand new PhD degree in Biochemistry, and Marty – as we all called him – had offered me to stay in his house until I found housing of my liking. I had come to Rodbell’s laboratory because he had recently published work on the isolation of free fat cells and their exquisite sensitivity to insulin, including that of insulin’s effect to antagonize the lipolytic actions of catabolic hormones such as epinephrine, glucagon and adrenocorticotropin, ACTH. Sutherland’s group had shown that these hormones induce lipolysis using cAMP as a second messenger (a discovery for which he was to receive the 1971 Nobel Prize in Medicine), and that in adipose tissue, insulin reduced the levels of cAMP that had been elevated by lipolytic hormones (shown for epinephrine in — adapted from Butcher et al., 1966 [1
]). It was Rodbell’s hypothesis that insulin antagonized lipolysis by inhibiting the adenylyl cyclase, and this would be my project in his laboratory. Knowing that I would have to measure adenylyl cyclase I had, before leaving Argentina, set up the assay used by Sutherland’s group to quantify cAMP. This assay used an enzymatic reaction cascade in which the cAMP formed by the enzyme stimulated phosphorylase-b
-kinase kinase, the activated phosphorylase-b
-kinase phosphorylated muscle phosphorylase b
and the activated phosphorylase (phosphorylase a
) converted glucose-1-phosphate to glycogen with formation of inorganic phosphate, which was visualized with a colorimetric Fiske–Subarow reaction. As sources of phosphorylase-b
-kinase and its kinase (now known as cAMP-dependent protein kinase or PKA), I used a crude glycogen preparation isolated from rabbit skeletal muscle which I knew came with glycogen metabolizing enzymes adsorbed to it. The phosphorylase b
in the assay came from a batch of rabbit muscle enzyme I had crystallized during my thesis work guided by Micheal Appleman, who was doing his postdoctoral work with Luis Leloir at the Institute for Biochemical Research “Fundación Campomar” in Buenos Aires. Leloir, who with Héctor Torres had co-signed my PhD thesis, directed the Campomar Foundation, and would later receive the 1970 Nobel Prize in Chemistry for the discovery of sugar nucleotides a few years before I had come to the Campomar laboratories. It is difficult to describe my surprise, when on that first day, Rodbell showed me a brand new assay for measuring adenylyl cyclase based on conversion of [α-32
P]ATP – 3 million cpm per assay tube; purchased from ICN – to [32
P]cAMP, that could be completed in an hour or so. The assay gave a blank of 150–200 cpm, a basal activity of 300–350 cpm and an ACTH-stimulated activity of 450–500 cpm. The method to isolate the [32
P]cAMP was easy, fast, and vastly superior to the Sutherland assay, and consisted of a two-step separation of the reaction product from the substrate and its breakdown products. This separation was based on Gopal Krishna’s observations that cAMP was retarded on a Dowex50-H+
column, eluting after the bulk of the unconverted [32
P]ATP and its hydrolytic products [32
P]ADP and [32
P]AMP, and that 32
P-labeled products that still contaminated the [32
P]cAMP in the Dowex 50 eluates could be adsorbed onto a nascent mixture of BaSO4
. The [32
P] cAMP formed by the enzyme was recovered in the supernatant after a short spin in a table top clinical centrifuge and quantified by liquid scintillation counting. For the next months I worked side by side with Martin Rodbell and his technician, Ann Butler, on the final validation steps of the assay, improving conditions, reducing variability and on increasing our assay capacity to more than the 20 columns that Rodbell was handling when I first came. The NIH Machine Shop made racks that held the Dowex columns in 5 rows of 10; we purchased a Sorval GLC-1 benchtop centrifuge in which we could spin the BaSO4
suspensions, 48 tubes at a time, and added higher levels of unlabeled ATP, theophylline, EDTA, and an ATP regenerating system to improve linearity of cAMP production as a function of time. Authentic [3
H]cAMP was added at the end of the assays to monitor recovery in the BaSO4
Fig. 1 Epinephrine increases and insulin opposes and reverses the epinephrine-induced increase in cAMP levels in isolated fat pads. (Adapted from Butcher et al., 1966 ).
Sometimes addition of insulin appeared to reduce stimulation of the fat cell ghost adenylyl cyclase by ACTH or epinephrine, but this was highly variable.
I had arrived on a stormy January 31st, 1967. On July 1st, Rodbell left for what turned out to be a 16-month sabbatical stay in Albert Renold’s Institute of Clinical Biochemistry at the University of Geneva, to investigate other effects of insulin on fat cells. Left alone with a solid training in enzymology from my graduate student time in Leloir’s institute, and a capacity to run up to 100-tube assays per day, I continued characterizing the adenylyl cyclase from an enzymological view point. A typical day started at 9 am with the arrival of 20 male Sprague–Dawley rats from which I dissected epididymal fat pads, which were digested in a medium with collagenase, to then prepare free fat cells by passing the digested tissue through silk screen and floating the cells away from remaining debris by a short 15-s spin in the benchtop centrifuge. I then suspended the fat cells in a hypotonic medium and lysed them by gently inverting the tubes with the cells 10 times, as Rodbell had taught me. Upon lysis the cells released their fat, as well as most of their contents, resealing immediately thereafter, and yielding sacs that Martin Rodbell called “fat cell ghosts”, and I often referred to as “anemic fat cells”. They had lost about 80–90% of their cytoplasmic content, retaining nuclei, some mitochondria, and an active hormone-sensitive adenylyl cyclase system. These membrane sacs were collected as a pellet in the benchtop centrifuge.
I generated time courses at low and high ATP, and pH curves, plus and minus insulin. I assayed activity at low and high Mg2+
, without and with EDTA in the assay, plus and minus insulin. I tested the effect of different monovalent cations at low and high Mg2+
, in the absence and presence of EDTA or EGTA, plus and minus insulin. I always tested for basal and ACTH- and fluoride-stimulated activities. Fluoride had been shown by Sutherland’s group to stimulate adenylyl cyclase activity by a mechanism equally obscure as the one by which hormones stimulated activity, but unrelated to fluoride’s ability to inhibit ATPases that hydrolyze the substrate. I discovered that EGTA, but not EDTA, abolished stimulation by ACTH and documented a requirement for Ca2+
in the action of ACTH, but not in that of glucagon or epinephrine, the other two hormones I was often testing for their ability to stimulate adenylyl cyclase. Sporadically there seemed to be a reduction in cAMP formed in the presence of insulin. But these effects were never reproducible. I wrote weekly letters to Rodbell — by hand, on thin airmail paper. Once I thought that I had it, and wrote that definitely there seemed to be an effect of insulin if I used very low Mg2+
, but that he should not tell anybody until I could confirm it better. He promptly mentioned it at that year’s Laurentian Hormone Conference – August 1967 – and it appears in the record of the discussion that followed his presentation. Marty made me a coauthor on his chapter for this contribution (Rodbell et al., 1968 [2
Spurred by ACTH’s requirement for Ca2+
, I wondered whether ACTH was acting on the same enzyme as epinephrine and glucagon. Sutherland’s group had proposed hormone-sensitive adenylyl cyclases to be a family of single molecules, each hormone interacting with its adenylyl cyclase (, adapted from Robison et al., 1967 [3
. If so, the effects of epinephrine and ACTH should have been additive. They were not. Moreover, propranolol, a beta-adrenergic receptor blocker, inhibited only the effect of epinephrine, leaving those of ACTH and glucagon untouched. It became clear that the hormone binding sites, the receptors, were separate from adenylyl cyclase, a theme we would explore in more detail after Rodbell’s return from Europe.
Fig. 2 Adenylyl cyclase as an epinephrine receptor model. (Adapted from Robison et al., 1967 ).
The relationship between ATP and Mg ion baffled me. High ATP was inhibitory at low Mg2+ but not at high Mg2+, high Mg2+ would stimulate absolute activity more in the absence than the presence of hormone. I eventually designed a matrix experiment, in which I varied both variables simultaneously. It took me about a week to plan the experiment and to calculate mixtures in which I varied both, unlabeled and radioactive ATP, so as to economize the use of the labeled compound. Labeled ATP was expensive — $240/mCi, and I must have used my weekly allowance of 5 mCi for this single experiment. Calculations were done by hand and slide rule —the programmable Wang calculator would arrive 2 years later; Neil Armstrong had not yet set foot on the Moon. The day before the assay, I numbered the 10×75 mm test tubes I would use, which I had washed the previous day by overnight submersion into chromosulfuric cleaning solution. In fact, I washed tubes every day, leaving them drying overnight for use on the next day, while the new batch was soaking in the red-brown sulfuric acid mix for rinsing the next day. Nowadays, disposable tubes are clean and can be used directly. The day of the matrix experiment, I started very early, pipetted the reaction mixes and placed them on ice before starting the fat cell-fat cell ghost preparation. Although normally I had no access to technical help, for this experiment, I had the help of Ann Butler: I started the reactions by adding 10-μl aliquots of ghost suspension to 40 μl of pre-pipetted mix and placed the tube into the 30 °C Dubnoff shaking incubation bath, and she stopped the reaction 10 min later with a “stop” solution containing cold ATP, carrier cAMP, labeled [3H]cAMP and sodium dodecyl-sulfate, all at 15 s intervals.
The results from the matrix experiment were important (). For one, they ordered and made sense of the many previous experiments where I had measured the ATP-dependence of the enzyme at one or another Mg2+ concentration, or measured the effect of varying Mg2+ at one or another concentration of ATP. Moreover, it gave me the first good insight into the fact that the mechanism by which fluoride and hormone stimulated activity should basically be the same, or if not, at least very similar. What I learned was that the dependence of activity on ATP followed pretty much Michealis–Menten kinetics with an apparent Km of 0.1–0.2 mM, when assayed in the absence of hormone or fluoride. In contrast, for both hormone- and fluoride-stimulated activities, the dependence on ATP showed some cooperativity and appeared not to saturate until above 3–5 mM. Hence, relative stimulations by hormone and fluoride were higher at high than at low ATP. The dependence on Mg2+ (in excess of ATP plus EDTA in the assay), was clearly cooperative for all three activities, basal, hormone- and fluoride-stimulated, with a Hill coefficient of 2 for all three, but differing in that hormone and fluoride activities were left-shifted along the Mg2+ concentration axis with respect to basal (). The activation mechanism, I concluded, was one of lowering the enzyme’s requirement for Mg2+. The fact that the apparent Km for ATP was right shifted for hormone and fluoride-stimulated activities with respect to basal, was a puzzle for which the explanation became clear only after we had learned about the GTP effect on glucagon binding.
Fig. 3 (a) The Matrix Experiment: Dependence of basal (middle panels), fluoride- (top panels), and ACTH- (bottom panels) stimulated adenylyl cyclase activities in fat cell ghosts on the concentrations of ATP (left panels) at the indicated MgCl2 concentrations, (more ...)
I did not run the full matrix experiment without and with insulin, but with the results at hand, I tested for an effect of insulin at critical points along the ATP–Mg2+ continuum, and eventually settled concluding that there was no effect. Now we know that the reduction of cAMP levels observed by Sutherland’s group had been due to activation of the phosphodiesterase — I had been looking at the wrong enzyme.
After the matrix experiment, I re-focused on the interdependence of the stimulation of the ghost adenylyl cyclase by hormones. From Mathys Staehelin at CIBA, Rodbell received an ACTH analog with four D-amino acids substituting for their corresponding L-amino acids. It proved to be a competitive inhibitor that did not affect stimulation by either glucagon or epinephrine. Later, we tested a glucagon homologue, des-His1-glucagon, given to us by Finn Sundby from the Novo Research Institute in Denmark, and found it to block the action of glucagon without affecting those of ACTH and epinephrine. It became clear to us that receptors had to be molecules that were separate from adenylyl cyclase. Fat cell adenylyl cyclase and lipolysis was also stimulated by LH, a glycoprotein hormone, and we proposed the model shown in : several hormone receptors affect a single adenylyl cyclase by changing its interaction with Mg2+.
Fig. 4 1969 conceptualization of an adenylyl cyclase system stimulated by multiple hormones, each interacting with a separate receptor having two domains: a ligand binding domain responsible for hormone specificity and a common adenylyl cyclase domain allowing (more ...)
Eventually, Martin Rodbell returned from his sabbatical in Europe. This had two consequences. I had an ear for my thoughts and the size of the group expanded to include Steve Pohl, a research associate, who arrived 2 months before Marty’s return, and Michiel Krans, a Dutch fellow, who arrived a few months after Rodbell. Tom Demar joined us as well to take care of us and make sure we had clean materials to work with. We also expanded from the single 200 sq. ft laboratory in which Rodbell and Ann Butler had shown me the adenylyl cyclase assay, to a second 200-sq. ft laboratory on the 8D corridor of Building 10. Steve helped me to bring the data on the kinetics of the fat cell adenylyl cyclase to a publishable form, and in February 1969 we submitted two papers to the J. Biol. Chem., one on the fat cell enzyme properties including the matrix experiment (Birnbaumer et al, 1969 [4
]), and the other on the existence of separate hormone receptors acting on a common adenylyl cyclase system (Birnbaumer and Rodbell, 1969 [5
]). When secretin, purified by Victor Mutt and Erik Jorpes at the Karolinska Institute in Stockholm, was shown to be lipoplytic, we tested its effect on the fat cell adenylyl cyclase and found it too to stimulate through a receptor site distinct form those mediating the effects of epinephrine, ACTH and glucagon (Rodbell et al., 1970a [6
We began thinking of a transduction mechanism and of Mg2+
as an integral part of this mechanism. We began speaking of discriminators, transducers and amplifiers (), which now correspond to GPCRs, G-proteins and effectors, but then were just concepts that facilitated communication. The role of GTP still had to be discovered and our thoughts were more on the side of phospholipids playing key roles in transduction, as different detergents had distinct and non-linear effects (Rodbell et al., 1970b [7
Fig. 5 Late 1969 representation of the hormone sensitive adenylyl cyclase as a system formed of three functional elements: Discriminator, Transducer and Amplifier. (From Rodbell et al., 1970b ).
In spite of the beauty of the adenylyl cyclase system in fat cell ghosts, it was not very amenable to biochemical study. Fat cell ghosts could not be frozen and thawed with preservation of hormone-stimulated adenylyl cyclase activity. This required us to prepare fresh fat cells every day. The original work by Sutherland and colleagues leading to the identification of cAMP as a second messenger had been done with dog liver homogenates, in which they found that glucagon and epinephrine were able to activate phosphorylase. They had gone on to show that the hormones interacted with the particulate fraction to generate a heat stable factor – cAMP –that then caused the activation of phosphorylase in the supernatant in which the hormones were unable to promote phosphorylase activation (Berthet et al., 1957 [8
]). From David Neville at the National Institute of Mental Health (NIMH), we obtained a highly purified preparation of liver plasma membranes which Steve Pohl tested for presence of epinephrine and glucagon responsive adenylyl cyclase. Even though the liver membrane adenylyl cyclase was not stimulated by epinephrine, it had a robust response to glucagon that was the same in fresh and frozen membranes (Pohl et al., 1969 [9
]). We taught Tom Demar to prepare Neville membranes, as we called them, and had him make industrial quantities that went into tubes stored in liquid nitrogen. This allowed us to pull out what we needed when we needed, without having to worry about their replenishment. Steve and I began studying the properties of the liver plasma membrane system and to establish similarities and differences between liver and fat cell adenylyl cyclases (Pohl et al., 1971 [10
]; Birnbaumer et al., 1971 [11
]). In parallel, Rodbell with Michiel Krans began to work on labeling glucagon with 125
I and to develop an assay to measure the binding of glucagon to liver membranes with the intention of characterizing the liver glucagon receptor.
At that time this was the logical next step in researching the mechanism of action of hormones, and we were not the only ones to take this course of action. In fact several studies of this type were being done quite near to us. But who was doing what, was not always known to me. I knew that Pierre Freychet, a visiting fellow from France with Jessie Roth, had labeled insulin with 125
I in a laboratory around the corner of ours and was validating an insulin receptor binding assay that laid the foundations of the present understanding of insulin’s interaction with its receptor (Freychet et al., 1971a [12
]; and b [13
]). But I did not know that Kevin Catt, who had experience in setting up radioimmunoassays, and hence in hormone labeling techniques, was beginning to study binding of 125
I-labeled hCG to testis membranes at the other end of Building 10 (De Kretser et al., 1971 [14
]; Catt et al., 1971 [15
]). I also did not know that Robert Lefkowitz, who worked in Ira Pastan’s laboratory and off and on used one of our two liquid scintillation counters — ‘Hi’ he would say ‘I am Bob Lefkowitz. I work in Ira Pastan’s lab. Can I use your scintillation counter tonight?’ — was working on an ACTH binding assay with adrenal membranes. Maybe this was because I was too encapsulated in my own world. This world had 1000% of my attention. Marty and Michiel, after several trials, had settled on labeling glucagon with 125
I using low concentrations of chloramine T as catalyst. I set up ISCO’s preparative polyacrylamide gel electrophoresis system to which we applied the full iodination reaction, containing 1 mCi of 125
I. I gave the fractions from the electrophoresis to Michiel and Marty to test for binding to liver membranes. Glucagon was very sensitive to damage by oxidation and the reaction mix had several labeled peaks, of which one bound to the membranes with the expected characteristics: specific for glucagon, as it was not prevented by excess ACTH or secretin, but was prevented by unlabeled glucagon. Moreover, the binding occurred over a concentration range similar to that over which glucagon stimulated the liver adenylyl cyclase activity (Rodbell et al., 1971a [16
]). The binding assay was simple: membranes were incubated with 125
I-labeled glucagon in the presence of 1% albumin, a Tris buffer, 1 mM EDTA, and additives such as unlabeled glucagon, ions, etc. The reaction was stopped by layering the reaction mixture on top of a 10% sucrose solution in a Beckman polyethylene microfuge tube, followed by a 5 min spin in the cold room — I remember Michiel rushing from lab to cold room (exactly across from the lab), starting the microfuge, hurrying back to the lab, where Marty was removing the next five samples from the incubation bath, overlaying them on sucrose and giving the tubes to Michiel for centrifugation. All done at 5 min intervals. The tube tips (bottoms) with the washed and pelleted membranes were then cut off with a scalpel and 125
I retained by the membranes was quantified in a gamma ray scintillation counter.
We thought we had a receptor assay, and presented some of our results at a colloquium on “The Role of Adenyl Cyclase and 3′5′-cyclic AMP in Biology”, held in November 1969 at the Fogarty Center on the NIH Campus (Rodbell et al., 1970b [7
]). At this same meeting, Bob Lefkowitz presented some of his data on preparation of iodinated ACTH and its interaction with adrenal membranes, published soon thereafter (Lefkowitz et al., 1970 [17
]). Before submitting our data for publication, we still performed a time course study which however gave an unexpected result. At 5 nM of 125
I-glucagon, the binding reaction took about 20 min to reach equilibrium. And, while addition of glucagon at time zero prevented 125
I-glucagon from binding, post-addition at 20 min resulted in very slow release of the labeled hormone (). Functional studies with the same membranes in which we stimulated adenylyl cyclase activity with 4–5 nM labeled or unlabeled glucagon, a concentration that caused an activation that was about 50% of maximal, gave time courses of cAMP accumulation, taken at 30 s intervals, that extrapolated to the time of glucagon addition () and did not show the lag-time predicted by the time course of “receptor” occupancy. Moreover, post-addition of des-His1
-glucagon, the competitive inhibitor of glucagon’s ability to activate adenylyl cyclase, stopped the activation by glucagon with at most a 30 s delay (), an effect that was predicted by the binding study to be quasi irreversible ().
The binding of 125I-glucagon to liver plasma membranes: Effect of GTP to accelerate the interaction rates. (Adapted from Rodbell et al., 1970c).
Fig. 7 Rapid activation of liver adenylyl cyclase by glucagon and its fast reversal. (From Birnbaumer et al., 1972 ).
Since there was a clear difference in the incubation conditions used for measuring adenylyl cyclase and those at which we were measuring glucagon binding, we decided to carry out the binding assay under adenylyl cyclase assay conditions, i.e., in the presence of ATP, MgCl2
and the ATP regenerating system (creatine kinase and creatine phosphate). The result was again unexpected: in a 15 min binding assay, addition of adenylyl cyclase assay reagents decreased the amount of 125
I-glucagon bound by about 50% (Rodbell et al., 1971b [18
]). Little did we know that we had just found the “GTP effect” and that this finding would be the beginning of the “signal transduction by G proteins” era. In fact the term signal transduction did not yet exist, though no doubt it would evolve from the transducer function we were proposing to intervene between hormone binding and adenylyl cyclase activation.
Of the reagents added, we rapidly learned that it was the ATP that reduced binding. As to specificity, GTP and ATP, but not UTP or CTP, were effective, and, to our surprise, GTP was effective at 1000 times lower concentrations than ATP. Among different guanine nucleotides, the only other form able to reduce binding was GDP (Rodbell et al., 1971b [18
]). Nelson Goldberg, who had developed an assay for cGMP based on its hydrolysis to 5′GMP and subsequent enzymatic conversion to GTP, and depended on the purity of ATP used to phosphorylate GMP to GTP, had recently reported that commercially available ATP was contaminated with GTP, varying from a low of 0.1% up to 5% and even 10%. It became clear therefore that I had never measured adenylyl cyclase activity without adding GTP. The possibility arose that GTP might be necessary for hormonal activation of adenylyl cyclase. This might be the reason that high levels of ATP were needed to observe robust hormonal stimulations relative to basal activity in the matrix experiment.
We split tasks. I looked for a functional correlate to the GTP effect on binding at the level of adenylyl cyclase activity, using the assay I had been using, while Marty Rodbell sought to develop an alternate substrate for adenylyl cyclase: AMP-PNP, now AMP-P(NH)P (Rodbell et al., 1971c [19
Rodbell synthesized [α-32P]AMP-P(NH)P from [α-32P]ATP and P(NH)P, which he obtained from Ralph Yount at Washington State University (Pulman, WA). Yount had shown that AMP-PNP could replace ATP in the allosteric regulation of the muscle actomyosin ATPase, without being a substrate for the ATPase. Rodbell’s idea was that if AMP-P(NH)P was a substrate for adenylyl cyclase he might be able to test for regulatory effects of GTP and ATP that might not be mimicked by AMP-P(NH)P.
On my side, I found what I already knew, i.e., that at very low ATP I could not obtain a robust stimulation by glucagon, and that addition of either ATP or (and this was new) GTP, led to robust stimulation of adenylyl cyclase by glucagon. Rodbell’s approach proved to be more definitive. Using an enzymatic approach based on the three-step synthesis of aminoacyl tRNAs, in which an intermediate of the form [amino acyl~tRNA~AMP] is formed from ATP and amino acid, that can decay to back to amino acid, tRNA and ATP, if excess PPi is added, he drove the intermediate to form AMP-P(NH)P by adding excess P(NH)P and made [α-32P]AMP-P(NH)P from [α-32P]ATP and P(NH)P. [α-32P]AMP-P(NH)P proved to be a substrate for adenylyl cyclase, which could now be assayed for hormonal stimulation under conditions that would not lead to transphosphorylations among GTP and ATP. Addition of glucagon still stimulated activity when added to the incubation at the same time as liver membranes. But, addition of glucagons 10–15 min after the cyclase reaction had been started, yielded no increase in activity unless GTP was also added ().
Fig. 8 Essential role for GTP in the activation of liver adenylyl cyclase by glucagon. (From Rodbell et al., 1991c ).
At the level of 125
I-glucagon binding, addition of GTP was found to accelerate both, dissociation and association. Binding was now reversible, which it was only very poorly in the its absence; addition of GTP to membranes with pre-bound 125
I-glucagon caused it to dissociate (). We also determined that the reduction of 125
I-glucagon-binding observed when we had added adenylyl cyclase assay reagents – now GTP –resulted from the fact that the rate of the dissociation was increased more than that of association, so that binding as a function of 125
I-glucagon concentration was right shifted by a factor of about 3- to 5-fold. Accordingly, at equilibrium the occupancy of the binding site was lower in the presence of GTP than in its absence ()2
Fig. 9 The effect of GTP to decrease the apparent affinity of the liver glucagon receptor for its ligand. (From Rodbell et al., 1971c ).
We speculated that actions of hormones could be regulated from within the cell and were not necessarily an obligatory consequence of simple changes in the hormone’s circulating levels. We began thinking of the transducer as being the site of action of GTP. In March 1970, we drew the model shown in , in which rapid binding of glucagon had a driving force operating on the transducer which then acted on adenylyl cyclase increasing its activity. Whether or not the GTP-binding transducer was a molecule separate from either receptor or adenylyl cyclase, was not addressed (Rodbell et al., 1970c [20
Late 1970 model of the glucagon-sensitive liver membrane adenylyl cyclase viewed as an information transfer system, incorporates the effects of GTP and ascribes a role for GTP in signal transduction.
Evidence for the existence of a separate molecule with a transducer function came from three quite independent research lines. Two were logical extensions of the line initiated by the GTP effects on glucagon binding and its role in glucagon receptor-mediated activation of the adenylyl cyclase system. The third, while prompted by the nucleotide requirement in hormonal activation of adenylyl cyclase, focused on biochemical changes occurring in rod photoreceptor cells upon illumination.