Inhibition of subunit dissociation and release of the stimulatory G-protein, G(s), by βγ-subunits and somatostatin in S49 lymphoma cell membranes

Abstract

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, G(s) and the inhibitory guanine-nucleotide-binding protein, G(i), in cell membranes of S49 lymphoma cells. In these cells, β-adrenergic receptors stimulate the activity of adenylate cyclase via G(s) whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, G(i). Using an antibody that selectively recognizes α(s), the monomeric, but not the heterotrimeric, α-subunit of G(s), we quantified the extent of dissociation of G(s) in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 μM-isoprenaline, 100 μM free Mg2+ and 100 μM-GTP produced substantial subunit dissociation of G(s), which was reversible by addition of purified βγ-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated G(s) (k(inhib.) for somatostatin = 51 ± 12 nM) and in the activity of adenylate cyclase (k(inhib.) = 121 ± 20 nM). By contrast, after addition of a 10-fold molar excess of βγ-dimer relative to α(s), there was a 2-3 min lag, after which the βγ-dimer re-associated G(s). Isoprenaline-induced dissociation of G(s) was accompanied by a release of α(s) from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of α(s) by the β-agonist and reversal by somatostatin. Membrane release of G(s) by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conlcude that in native cell membranes somatostatin-induced activation of G(i) dissociates G(i) and interferes with the G(s) activation cycle by providing βγ-dimer, which acts to prevent or reverse formation of monomeric α(s). Because α(s) can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated α(s) is likely to be an important factor in modulating the activity of adenylate cyclase.