GTP BINDING PROTEINS AND ADENYLATE CYCLASE

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Adenylate cyclase systems consist of inhibitory and stimulatory receptors linked to a catalytic unit through inhibitory and stimulatory proteins, Gi and Gs, respectively. Similarly, the retinal photon receptor rhodopsin is linked with a phosphodiesterase through transducin (T), which is similar to Gs and Gi. Each has a guanyl nucleotide-binding Alpha subunit (45 kDa Gs, 41 kDa Gi, and 39 kDa T) and Beta, Gamma subunit (35 and l0 kDa). Gs and Gi can be activated by NaF with AlCl3 and by decavadanate, as well as by agonists acting through specific receptors. The effects of NaF with AlCl3 and decavanadate on transducin were investigated in a reconstituted system consisting of the purified subunits of transducin (TAlpha, TBeta Gamma) and rhodopsin in prhosphatidylcholine vesicles. NaF noncompetitively inhibited the GTPase activity of TAlpha in a concentration- and time-dependent manner. Inhibition by NaF was enhanced synergistically by AlCl3 which alone only slighly inhibited GTPase activity. None of the other anions tested reproduced the effect of fluoride. Fluoride inhibited [3H]GppNHp binding to TAlpha and release of bound GDP. Vanadate (decameric) inhibited [3H]GTP binding to TAlpha and GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP. The ADP-ribosylation of TAlpha by pertussis toxin and binding of TAlpha to rhodopsin, both of which are enhanced in the presence of TBetaGamma, were inhibited by NaF with AlCl3 and by vanadate. These findings are consistent with the conclusion that NaF plus AlCl3 or vanadate can cause the dissociation of TAlpha from TBetaGamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation and inhibition presumably result from similar effects of NaF plus AlCl3 and vanadate on Gs and Gi.