Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system.
Insulin stimulates phosphorylation of both alpha- and beta- subunits of its own receptor in a cell-free system. A solubilized lectin-purified preparation of insulin receptors from rat liver membranes was preincubated with or without insulin at 4 degrees C and labeled for 10 min with Mn[gamma- 32P]ATP; the receptor subunits were isolated by specific immunoprecipitation with anti-receptor antibodies, followed by gel electrophoresis in sodium dodecyl sulfate. In gels run under reduced conditions, two bands (Mr = 135,000 and 95,000) were selectively labeled. These correspond exactly to the position of the alpha- and beta-subunits of the insulin receptor. Labeling of the Mr = 95,000 band was approximately 5-fold that of the Mr = 135,000 band. No labeled bands were detected when identical samples were immunoprecipitated in control serum. Phosphorylation of the receptor subunits required the presence of the divalent cation Mn2+ or Co2+; other cations such as Mg2+, Cr3+, Ca2+, and Zn2+ were ineffective. [gamma- 32P]ATP served as the 32P donor, whereas [gamma- 32P]GTP was ineffective. Phosphorylation of both subunits was stimulated 4-6-fold after a 60-min exposure to 10(-7) M pork insulin. Insulin-stimulated phosphorylation was half-maximal after 5 min of incubation with 10(-7) M insulin or after 18 h with 3 X 10(-10) M hormone. The enhanced phosphorylation was specific for insulin and its analogs; guinea pig insulin was about 2% as potent as pork insulin, whereas epidermal growth factor, adrenocorticotropic hormone, and glucagon, as well as cAMP, were ineffective. The rapidity and specificity of this reaction, as well as the presence of all necessary components in the plasma membrane, suggest that insulin-mediated receptor phosphorylation is one of the earliest biochemical steps following insulin binding.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D007328:Insulin; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin