Phosphorylation of G-protein α-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects

Abstract The phosphorylation of G-protein α-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [ 32 P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein α-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as G s and G i α-subunit, respectively, by comparison with [ 32 P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [ 32 P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [ 32 P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with G α s , since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the G s α-subunit, the phosphorprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the G s α-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein α-subunits in the action of insulin.