Changes in the phosphorylation state of the inhibitory guanine‐nucleotide‐binding protein G_i‐2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats

Abstract

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

Phosphorylation of Gzα by Protein Kinase C Blocks Interaction with the βγ Complex

“…Houslay and colleagues (35) identified the specific G protein subtype phosphorylated in this system as G i␣2 , and several groups observed a correlation between phosphorylation of G i␣2 and inactivation of signaling through this G protein (36 -38). Further, they observed that treatment with a phosphatase restored signaling through G i␣2 (37,39). Our model offers a possible biochemical explanation for their observations, in that phosphorylation of G i␣2 could also interfere with its subunit reassociation.…”

Differences in Binding of Hepatic Nuclear Proteins from Lean and Obese Rats to the 5‘‐Upstream Region of Tyrosine Aminotransferase

“…This could result either from decreased activity of a specific kinase or increased phosphatase activity in the obese rat. Houslay and colleagues (7,44) have previously suggested that altered phosphorylation was responsible for the loss of Gi action on adenyl cyclase in hepatic membranes of obese rats. Further, it is recognized that the activities of many of the proteins that bind to this upstream region of TAT DNA (GR, Fos, Jun, HNF) are affected by phosphorylation (26,32,35).…”

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum

“…Detection of the G-protein, a-Gi-2, was done as described before [35,36] using the antiserum SG1 which was raised against the C-terminal decapeptide of the a-subunit of transducin (conjugated to KLH).…”