Biosynthetic labeling of insulin receptor: studies of subunits in cultured human IM-9 lymphocytes.

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Abstract
We have identified the subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) by biosynthetic labeling with [35S]methionine and specific precipitation with autoantibodies against the insulin receptor. IM-9 lymphocytes were cultured with [35S]methionine and extracted with Triton X-100. Insulin receptors were concentrated and purified 20-fold by chromatography of the cell extract on wheat germ agglutinin-agarose, and then specifically precipitated by receptor antibodies after addition of a second antibody. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed specific precipitation of two major bands with molecular weights of 130,000 and 90,000. Both species were precipitated by receptor antibodies from four different patients with the syndrome of extreme insulin resistance and acanthosis nigricans. In accord with previous data that insulin bound to receptor reduces the affinity of receptor for anti-receptor antibody, we found that preincubation of the wheat germ-purified cell extract with insulin (1.7 microM) prior to immunoprecipitation caused a decrease in the appearance of both species. The decrease in insulin binding seen after incubation of the lymphocytes with insulin for 12 hr ("down regulation") was associated with a decrease in the labeling of both the 130,000 and 90,000 bands. The apparent molecular weight of both subunits was decreased after pretreatment with mixed glycosidases. In conclusion, we have biosynthetically labeled the insulin receptor with [35S]methionine and showed that the receptor consists of two major glycoprotein subunits with apparent molecular weights of 130,000 and 90,000.