TRANSDUCIN GTPASE--GENES FOR GTP-BINDING PROTEINS

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Guanine nucleotide-binding proteins (GNP) are critical to the regulation of cellular metabolism. Three GNP's, Gs, Gi, and transducin, composed of Alpha, Beta, and Gamma subunits mediate hormonal stimulation of adenylate cyclase, hormonal inhibition of adenylate cyclase, and light activation of phosphodiesterase of retinal rod outer segments, respectively, A fourth GNP, Go, interacts with inhibitory hormone receptors, but does not appear to affect cyclase activity. A fifth GNP, p21, is a product of a proto-oncogene involved in the regulation of cell growth. To define the molecular mechanisms by which these GNPs couple external stimuli to intracellular events monoclonal and polyclonal antibodies were prepared and cDNA clones were isolated for some of these proteins. Three monoclonal antibodies prepared to purified transducin (T) reacted with TAlpha but not TBetaGamma and inhibited the ability of TAlpha in the presence of rhodopsin and TBetaGamma to hydrolyze GTP. Another monoclonal antibody against TAlpha cross-reacted with GiAlpha, GoAlpha and p21. All 4 antibodies reacted with the amino-terminal region of transducin. These studies are consistent with the presence of common epitopes in GNPs. Immunization of rabbits with TBetaGamma resulted in the production of anti-idiotypic antibodies against rhodopsin, consistent with the hypothesis that TBetaGamma binds to rhodopsin. To define further the regulation of transducin, a cDNA clone for the Gamma subunit was isolated from a Lambdagt10 cDNA library. Using the cDNA clone a single size class of message was demonstrated in total retinal RNA by Northern blotting; no homologous messages were detected in either brain or liver RNA. Polyclonal antibodies to TGamma did not cross-react with Gamma proteins from brain or liver. Thus, Gamma subunits appear to be tissue-specific.