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Open Forum on Cell Signaling

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ASBMB-ASPET Symposium: The G-Whizards of GPCR/G-Protein Signaling

13 May 2008

John F. Foley

Lee E. Limbird, Meharry Medical College, gave a stimulating historical overview of how the field of G proteins and G protein-coupled receptors (GPCRs) has evolved. She began by discussing the importance of the work of Earl W. Sutherland in characterizing cyclic adenosine monophosphate (cAMP) as a second messenger in hormone signaling, which essentially launched the field. Also important was the work of Martin Rodbell on the effect of guanosine triphosphate (GTP) on cAMP synthesis. This discovery was followed by experiments that showed that the hormone receptors and cyclases (enzymes that synthesized cAMP) in this system were separate molecules. The work of Alfred G. Gilman established that another protein, now known as a G protein, conveyed the effect of GTP on this system. The relationships between these three components (receptor, G protein, and cyclase) then had to be characterized in terms of their physical and functional interactions. Whereas agonists stabilized the association between the receptor and the G protein, GTP had the reverse effect. Thomas Pfeuffer then showed that GTP, but not GDP, stabilized interactions between the G protein and the cyclase. Functional interactions between the receptor and G protein were appreciated when it was shown that catecholamines, such as epinephrine, stimulated GTPase activity in turkey erythrocyte membranes. In terms of signals going back the other way, guanine nucleotides decreased receptor affinity for hormones and agonist drugs. Competition binding studies revealed that GTP regulated interactions between the agonist-bound receptor and the G protein. This was the beginning of the use of models to describe what was going on in this system, perhaps the most famous of which is the ternary complex model, which helps describe how agonists and GTP regulate the GPCR-G protein-cyclase system. Of course, as studies of other GPCRs, G proteins, and effector molecules have progressed, models have continued to change to address such concepts as the pre-coupled receptor and inverse agonism, in which a ligand binds to a constitutively-active receptor and inhibits its activity. This talk nicely provided the historical background for the other speakers in this session.

Robert J. Lefkowitz, Duke University Medical Center, spoke about the characterization of the structures and functions of GPCRs. Initial work in this field was hampered by the difficulty in isolating β-receptors; the key was getting the right high-affinity chromatography supports to enable receptor purification. In the 1980s, β-receptors were reconstituted in vesicles, which could be fused with cells that had no β-receptors, to restore responsiveness. This work led to the cloning, in 1986, of the β2-adrenergic receptor (β2-AR). What was shocking at the time was that the sequence of the β2-AR showed that it contained 7 transmembrane spans and had some homology with rhodopsin; it was thought that 7 spans must be a characteristic of light-sensitive proteins, such as bacteriorhodopsin. Now of course we know that this is a general feature of GPCRs. The cloning of the β2-AR was followed up with the cloning of the α2-ARs. These experiments thus established the general structure of the GPCRs. Further work by Dr. Lefkowitz and others has shown that the activation and desensitization mechanisms of all of the various GPCRs are also very well conserved. It was the study of the phosphorylation and desensitization of stimulated receptors that led to the isolation of β-adrenergic receptor kinase (βARK), the founding member of the family of GPCR kinases (GRKs), which now has seven members. These researchers then found that the more purified the preparation of GRK was, the less able it was to desensitize the isolated β2-AR. So some important component of this system was being lost, but what? The sequence of visual arrestin was used to help search for the missing components, which turned out to be the β-arrestins 1 and 2. Reconstitution studies then showed that the β-arrestins were very potent at desensitizing β2-ARs. There are now 4 members in the arrestin family. However, recent work has changed our way of thinking about how the β-arrestins work. Not only are they involved in desensitizing GPCRs (by binding to the cytoplasmic tails of GPCRs thereby blocking their interactions with G proteins), β-arrestins also serve as adaptor proteins during clathrin-mediated endocytosis of GPCRs. Furthermore, β-arrestins mediate signaling in their own right, for example in the activation of mitogen-activated protein kinases (MAPKs). β-arrestins are also involved in such processes as cell survival and chemotaxis, and act as scaffold proteins to bring together different members of the MAPK cascade.

Heidi E. Hamm, Vanderbilt University Medial Center, spoke on the interactions between GPCRs and G proteins and on the mechanisms of G protein activation. Dr. Hamm started her own research in the field of visual transduction. At the time, there was controversy as to the identity of the second messenger involved. Was it calcium, as suggested by electrophysiologists, or was it cGMP, as put forward by biochemists? The purpose of Dr. Hamm’s research was to try to use monoclonal antibodies to block the activation of the G protein transducin by light. She found such an antibody that also blocked the cGMP response. Peptide mapping of the epitopes recognized by the various antibodies that were studied revealed one peptide that blocked the receptor independently of the transducin. These studies and work with Paul Sigler and Joseph Noel led to solving the structure of the G protein α-subunit, transducin in 1993. Over the next few years, the structures of the GTPase domain and the helical loop were published and then studies continued to characterize the GTP switch region of the G protein. At about the same time, Al Gilman’s group worked on α-subunits and also on βγ dimers. In 1996 the crystal structure of the heterotrimeric G protein was published. All of these studies showed that the GTPase domain and the N-terminal region of the α-subunit have interactions with the β-subunit, but that there are no contacts between the α and γ-subunits. In 1998 it was shown that residues of the βγ-dimer that were involved in interactions with the α-subunit were also important for interactions between the βγ-dimers and effector molecules. Dr. Hamm emphasized how important the C-terminal region of the α-subunit of the G protein is, as it is through this region that the α-subunit binds to the GPCR and stabilizes its high-affinity binding state. The work of Henry R. Bourne and Bruce R. Conklin on chimeric G proteins showed that the last five amino acid residues of the C-terminus of a G protein α-subunit are critical for GPCR-coupling specificity. Dr. Hamm then outlined more recent research that has helped to understand how activation of the GPCR triggers binding to the G protein. Future challenges include determining what the GPCR-G protein complex really looks like and to find out how the GPCR catalyzes the release of GDP from the G protein α-subunit.

Alfred G. Gilman, University of Texas Southwestern Medical School, started his MD PhD program in 1962 in Earl W. Sutherland’s laboratory, where he worked on adenylyl cyclase (AC); cAMP having been discovered in 1957. At the time, it was thought that hormones could not work on anything other than intact cells; a "rule" that Sutherland successfully broke. In his talk, Dr. Gilman also presented an historical perspective on how AC and G proteins were discovered; the AC being one of the last pieces of the puzzle to be completed. At the end of his talk, Dr. Gilman looked to the future of research in this field. It seems likely that not so many more new components are likely to be discovered in this system, so now the challenge is to find people who are willing to do the really hard work of fully understanding the mechanisms involved in GPCR-G protein interactions and signaling. Most of our knowledge has come from relatively simple experimental conditions with a single ligand-receptor pair, but it is unclear what really happens when multiple ligands are present and when many interactions are happening in the cell simultaneously. Dr. Gilman emphasized that imaging will be a very important technique to gain information about spatial and temporal dynamics. Another area of concern was how to explain why heterogeneous effects that are observed in a single population of cells may not have correlations within individual cells. In the future, researchers will need to adopt a more quantitative approach and to look more at the level of the single cell.

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