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.