The effects of N-ethylmaleimide on the phosphorylation and aggregation of insulin receptors in the isolated plasma membranes of 3T3-F442A adipocytes.
We have examined the insulin-dependent phosphorylation of the insulin receptor in the isolated plasma membranes of 3T3-F442A adipocytes. Phosphorylation of the insulin receptor is detected readily in the plasma membrane of these cells by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulfate and under nonreducing conditions, the predominant species of phosphorylated insulin receptor has an apparent molecular mass of 350,000 daltons with the beta-subunit (92,000 daltons) being phosphorylated. The phosphorylation of the insulin receptor in the isolated plasma membrane is absolutely dependent on insulin; tyrosine residues and, to a lesser extent, serine residues of the receptor are phosphorylated. Treatment of the plasma membrane with N-ethylmaleimide (NEM) has two effects; 1) NEM prevents the formation of a larger form (greater than 350,000 daltons) of the phosphorylated insulin receptor. The formation of this larger form of the receptor involves sulfhydryl oxidation which occurs at 37 degrees C under nondenaturing conditions, but does not occur at 0 degrees C or at room temperature even in the presence of sodium dodecyl sulfate. These results indicate that the larger form of the phosphorylated receptor can occur under physiological conditions and suggest that this process may be relevant to aggregation of the receptor-ligand complex. 2) Prior to treatment with insulin, NEM enhances the phosphorylation of the insulin receptor. Phosphopeptide analysis indicates that the site(s) of phosphorylation of the receptor is identical in the presence or absence of NEM.
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011940:Receptor Aggregation / Q000187:drug effects*; D011972:Receptor, Insulin / Q000378:metabolism*; D013696:Temperature
D007328:Insulin; D011972:Receptor, Insulin; D005033:Ethylmaleimide