REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

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1) Regulation of the hormone-sensitive adenylate cyclase is mediated by two guanine nucleotide-binding (G) proteins, stimulation through Gs, inhibition through Gi. In the visual excitation system, a similar G protein, Gt or transducin, couples the photon receptor rhodopsin to a cyclic GMP phosphodiesterase. These G proteins are heterotrimers of alpha, beta, and gamma subunits. The alpha subunits bind guanine nucleotide and hydrolyze GTP. The beta gamma subunits facilitate coupling of alpha to receptor. To determine the function of G gamma, a mouse anti-Gt gamma monoclonal antibody (2H3) was prepared. 2H3 was specific for Gt gamma and did not recognize G gamma from liver or brain. Interaction of 2H3 with Gt alpha beta gamma appeared to facilitate the dissociation of the G protein into its alpha and Beta gamma components, as monitored by pertussis toxin-catalyzed ADP-ribosylation (which favors the heterotrimer and is inhibited by 2H3) and immunoprecipitation (in which Beta gamma was precipitated but not alpha). In the presence of receptor, the effect of 2H3 was diminished, consistent with the hypothesis that a domain on Gt gamma is masked in the rhodopsin Gt complex. The studies, thus, support a role of G gamma in a protein-receptor coupling. 2) Bacterial toxins, such as pertussis and cholera toxins, exert their effects of cells through the ADP- ribosylation of G proteins; this modification leads to altered function. In the case of cholera toxin, ADP-ribosylation of Gs alpha results in adenylate cyclase activation. A novel toxin from Escherichia coli (LT-II) appeared to catalyze the ADP-ribosylation of Gs alpha, leading to activation of adenylate cyclase, suggesting that LT-II and cholera toxin, two nonhomologous and immunologically different toxins, share a common mechanism of action.