Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes

Abstract

We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.