Insulin induces an unmasking of the carboxyl terminus of G(i) proteins in rat adipocytes.
Several groups have shown a relationship between the insulin receptor and inhibitory G proteins, G(i). An antisera, 8729, to a peptide sequence (KNNLKDCGLF) corresponding to the carboxyl termini of G(i)alpha subunits was used to investigate this relationship by immunoelectron microscopy. Rat adipocytes were incubated in the absence or presence of 100 ng/ml insulin for 1 h and fixed for immunoelectron microscopy. Insulin-treated adipocytes stained with 8729 were labeled at the cell surface at a much higher density than control adipocytes. Subcellular fractionation of insulin-treated and control cells was followed by PAGE and Western blots of the plasma membrane and low-density microsomes with 8729. The density of the bands did not change in response to insulin treatment. Antibodies to noncarboxyl terminus sequences of the alpha subunit were used for immunoelectron microscopy and no difference was noted between insulin-treated and control adipocytes. These results indicated that 8729 was detecting a conformational change in the structure of G(i)alpha subunit in the plasma membrane in response to insulin. This unmasking of the carboxyl terminus was also seen in response to treatment with phenylisopropyladenosine and prostaglandin E2. Pertussis toxin-catalyzed ADP ribosylation also unmasked the carboxyl terminus. In contrast, isoproterenol, an agonist of stimulatory G proteins (Gs), did not induce an unmasking of the carboxyl terminus. These results support the hypothesis that some of insulin's effects are mediated through G(i) proteins in adipocytes.
Record
R D
RD
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Smith
R M
RM
Jarett
L
L
D000273:Adipose Tissue / Q000737:chemistry* / Q000166:cytology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007106:Immune Sera; D007328:Insulin; D010566:Virulence Factors, Bordetella; D010660:Phenylisopropyladenosine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins