G proteins and dual control of adenylate cyclase

Recent studies have revealed that the hormone-sensitive adenylate cyclase system is far more complicated than originally suspected-consisting of at least three types of proteins embedded in the lipids of the plasma membrane. This complex of proteins has the ability to receive information from several sources and generate a single, integrated response. The decision-making process begins when receptors for a large number of hormones, neurotransmitters, and other regulatory molecules interact with appropriate endogenous ligands and with drugs at the cell surface. These interactions ultimately result in stimulation or inhibition of adenylate cyclase activity; there are subsequent alterations of intracellular phosphorylation as a consequence of the actions of CAMP-dependent protein krnases and counter-regulatory phosphoprotein phosphatases. Stimulatory receptors include those for P-adrenergic agonists, ACTH, gonadotropins, and many others, while Inhibitory control is exerted by such agents as a*-adrenergic and muscarinic agonists and opioids. Receptors communicate with a pair of homologous guanine-nucleotide-brnding regulatory proteins-one (G,) mediates stimulation of adenylate cyclase activity, while the other (G,) is responsible for inhibition. The G proteins control the activity of the actual catalyst of the enzyme system (C) in a complex series of reactions discussed below. Notable in this scheme is the fact that the two G proteins share a common subunit, and the action of this subunit appears to be crucial for the integrative capacity of the system. The G proteins that regulate adenylate cyclase activity are members of a larger family of homologous guanine-nucleotide-binding proteins that includes transducin, a regulatory protein of the outer segment of the retinal rod. The products of the ras genes may also be relatrves. Progress in dissection of the hormone-sensitive adenylate cyclase system was slow through the 1960s and much of the 1970s for a number of valid, and for some time unsuspected, reasons. These include the hydrophobic nature of the components: their multiplicity, lability, and extremely low concentration (1 part in 1 O5 of cell protein is typrcal); and the need for proper orientation of the components in an appropriate membrane for hormonal regulation of enzymatic activity. However, a number of advances have now led to a successful path of experimentation. It is clear that detailed understanding of the mechanism of regulation of CAMP synthesis will soon be achieved from study of the interactions of purified components that have been reconstituted in lipid bilayers of defined composition. G. and G, The two guanine-nucleotide-binding regulatory components of the adenylate cyclase system have been purified and studied in some detail-particularly in detergent-containing solutions (Sternweis et al., JBC 256, 11517-l 1526, 1981; Bokoch et al., JBC 258, 2072-2075, 1983). The table and two figures are included to facilitate the following discussron. The catalyst of adenylate cyclase is essentially inactive with its physiological substrate, MgATP, in the absence of G,. G, is thus most conveniently assayed by its ability to stimulate adenylate cyclase activity; the best source of C for such an assay is obviously one that is free of G,. Such resolved preparations of C can be made biochemically. Alternatively, the membrane of the cycS49 lymphoma cell mutant IS a superb assay vehicle. This mutant is deficient in G, activity, but retains C, and was particularly useful for elucidating the importance and activity of G,. It was originally assumed that cycwas devoid of adenylate cyclase, since it had essentially undetectable levels of this enzymatic activity. However, addition of G, to cycmembranes fully restores adenylate cyclase activity stimulated by hormone, guanine nucleotide, and fluoride. G, has an oligomeric structure with 45,000 and 35,000 dalton subunits, while G, has a similar structure with 41,000 and 35,000 dalton subunits. A third subunit, M, 10,000, also appears to be present in both proteins, although this has not been proved rigorously. The larger (a) subunit of each protein contains a site for NAD-dependent ADPribosylation catalyzed by a bacterial toxin. Cholera toxin ADP-ribosylates G,,, in the presence of a membrane-bound protein cofactor (ARF), while islet-activating protein (IAP; one of the toxins of Bordetella pertussis) ADP-ribosylates G,.,. Such ADP-ribosylation results in characteristic modificatlons of the function of each regulatory protein. The (Y subunits of each G protein also contain a site with highaffinity for guanine-nucleotide binding. The 35,000 dalton (@) subunits of G, and G, are indistinguishable from each other functionally (see below) and by analysis of amino acid composition and maps of proteolytic peptides. Incubation of either G, or G, with nonhydrolyzable guanine nucleotides (e.g. GTPyS) or with fluoride (in the presence of Mg2+ and A$+; see Sternweis and Gilman, PNAS 79, 4888-4891, 1982) results in “activation” of the protein. By activation I mean a state in which the protein is capable of either stimulating or inhibiting the activity of C. In fact, activation of either G, or G, by GTPyS is essentially irreversible (in the presence of Mg’+); free GTPrS can be removed, and the activated state is stable. Activation of either G, or G, has been shown to be the result of or associated with ligand-promoted dissociation of the protein’s subunits, as follows (Northup et al., JBC 258, 11369-I 1376, 1983; Katada et al., JBC, in press):