G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-triphosphate

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-triphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of M(r) 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-M(r) peptide is the α-subunit of the G-protein, referred to as G(i), that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-M(r) substrate for pertussis toxin was investigated. The fat-cell 39,000-M(r) peptide was compared structurally and immunologically with the α-subunits of two other G-proteins, G(t) isolated from the rod outer segments of bovine retina and G(0) isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [³²P]NAD⁺, the electrophoretic mobilities of the fat-cell 39,000-M(r) peptide and the α-subunits of G(0) and G(t) were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-M(r) peptide and the α-subunit of G(o). Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified G(t), G(i), G(o), and fat-cell membrane proteins. Although recognizing the 36,000-M(r) β-subunit band of G(t), G(i), G(o) and a 36,000-M(r) fat-cell peptide, antisera raised against G(t) failed to recognize either the 39,000- or the 41,000-M(r) peptides of fat-cells or the α-subunits of G(o) and G(i). Antisera raised against the α-subunit of G(o), in contrast, recognized the 39,000-M(r) peptide of rat fat-cells, but not the α-subunit of either G(i) or G(t). These data establish the identity of G(o), in addition to G(i), in fat-cell membranes and suggest the possibility that either G(o) or G(i) alone, or both, may mediate hormonal regulation of adenylate cyclase phospholipase C.