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

Abstract

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta 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 Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-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 (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-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 alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)