The inhibitory guanine nucleotide-binding regulatory compound of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc^- and wild type membranes

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (G(i) and G(s)) of adenylate cyclase both have an α·β subunit structure, and the β subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of G(i). Such inhibition can be explained by reduction of the concentration of the free α subunit of G(s) as a result of its interaction with the β subunit of G(i) in normal G(s)-containing membranes. However, inhibition in S49 lymphoma cyc^- cell membranes presumably cannot be explained by the G(i)-G(s) interaction, since the activity of the 2a subunit of G(s) is not detectable in this variant. Several characteristics of G(i)-mediated inhibition of adenylate cyclase have been studied in both S49 cyc^- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc^- and wild type membranes. (1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. (2). The effect of guanosine-5'-(3-O-thio)triphosphate (GTPγS) is essentially irreversible, and somatostatin accelerates GTPγS-induced inhibition. (3) Inhibition of adenylate cyclase by somatostatin or GPP(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). (4) Both cyc^- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the α subunit of G(i). (5) β Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTPγS. The α subunit of G(i) in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTPγS-bound form. The resolved subunits of G(i) have been tested as regulator of cyc^- and wild type adenylate cyclase under a variety of conditions. The α subunit of G(i) inhibits forskolin-stimulated adenylate cyclase activity in cyc^-, while the β subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTPγS) and the α subunit of G(i) are not additive in cyc^- membranes. In wild type, the inhibitory effects of the hormone and GTPγS are not additive with those of the β subunit. Inhibition caused by GTP plus somatostatin or that produced by GTPγS is relieved by the β subunit in cyc^- membranes, while the α subunit of G(i) relieves such inhibition in wild type. Reconstitution of cyc^- with G(s) restores the polarities of the effects of the G(i) subunits. The results of this study suggest that dissociation of the G(i) dimer is responsible for inhibitory regulation of adenylate cyclase in both S49 cyc^- and wild type membranes. In cyc^- membranes, a major fraction of the inhibition caused by guanine nucleotide and somatostatin appears to result from the action of the α subunit of G(i). The β subunit appears to mediate inhibition in wild type membranes, which contain functional G(s).