Phosphorylation of G-protein alpha-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects.
The phosphorylation of G-protein alpha-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [32P]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 alpha-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as Gs and Gi alpha-subunit, respectively, by comparison with [32P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [32P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [32P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with G alpha 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 Gs alpha-subunit, the phosphoprotein 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 Gs alpha-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 alpha-subunits in the action of insulin.
Joost
H G
HG
Institut für Pharmakologie, Universität Göttingen, F.R.G.
Schmitz-Salue
C
C
Hinsch
K D
KD
Schultz
G
G
Rosenthal
W
W
D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007328:Insulin / Q000502:physiology*; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism
D007328:Insulin; D019204:GTP-Binding Proteins