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  • Title: G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate.
    Author: Rapiejko PJ, Northup JK, Evans T, Brown JE, Malbon CC.
    Journal: Biochem J; 1986 Nov 15; 240(1):35-40. PubMed ID: 3103610.
    Abstract:
    Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt 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-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.
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