Changes in the phosphorylation state of the inhibitory guanine-nucleotide-binding protein Gi-2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats.
Treatment of intact, 32Pi-labelled hepatocytes from lean Zucker rats with a range of agents including 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the alpha-subunit of the inhibitory G protein of adenylate cyclase (alpha Gi-2). These agonist-induced phosphorylations of alpha Gi-2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5'-[beta,gamma imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist-induced phosphorylation of alpha Gi-2 and to p[NH]ppG-mediated inhibition of adenylate cyclase. The basal level of alpha Gi-2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 +/- 0.09 mol phosphate/mol alpha Gi-2 than in hepatocytes from lean animals which gave 0.54 +/- 0.09 mol phosphate/mol alpha Gi-2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of alpha Gi-2 in the hepatocytes from lean animals but had little effect on the phosphorylation of alpha Gi-2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of alpha Gi-2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of alpha Gi-2. The resistance to protein-kinase-C-mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine-nucleotide-mediated 'Gi function' seen in obese Zucker rats may be due to an inactivating phosphorylation of alpha Gi-2.
Bushfield
M
M
Department of Biochemistry, University of Glasgow, Scotland.
Pyne
N J
NJ
Houslay
M D
MD
D000262:Adenylyl Cyclases / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011924:Rats, Zucker / Q000378:metabolism*; D012016:Reference Values; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D010710:Phosphates; D014667:Vasopressins; D000804:Angiotensin II; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate