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

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.

Protein kinase activity of the insulin receptor

“…First, non-immune serum did not precipitate a band with a similar electrophoretic mobility. Second, the molecular size was identical with that previously determined using biosynthetic, cell surface, and affinity labelling methods (Jacobs et al, 1979;Van Obberghen et al, 1981;Massague et al, 1981). Subsequently, insulin-stimulated phosphorylation of the f-subunit of insulin receptor was demonstrated in cell-free systems using [y-32P]ATP in solubilized and partially purified receptor preparations from rat liver ( Fig.…”

“…2). The model is based on application of biosynthetic, cell-surface or affinity labelling of the receptor (Yip et al, 1978;Jacobs et al, 1979;Van Obberghen et al, 1981;Pilch & Czech, 1979;Siegel et al, 1981). With affinity labelling the a-subunit is labelled predominantly by radioactive insulin when compared with the fl-subunit, the labelling of which is much weaker (Massague et al, 1981), or even absent (Jacobs et al, 1979).…”

“…Finally, insulin receptors in human IM-9 lymphoblasts show different characteristics in comparison to those in liver and fat cells regarding faster association and dissociation kinetics of receptor-bound insulin, complete absence of receptor-mediated insulin degradation and a greater Mr of a-and fl-subunits (Sonne & Gliemann, 1980;Van Obberghen et al, 1981). The observed differences may indicate that the insulin receptors of transformed cell lines in culture generally have greatly altered properties which may be linked to a different role of insulin in these cells.…”

From receptor to transporter: insulin signalling to glucose transport

“…1) begin at the insulin receptor, an a 2b2 tetramer [9]. The a -subunit is located entirely at the extracellular face of the plasma membrane and contains the insulin binding site.…”

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells

“…Structurally, insulin receptors consist of a subunits of Mr 135000 and , subunits of Mr 95000 linked together through disulphide bonding to form tetramers (aAx/) (Massague et al, 1980;Van Obberghen et al, 1981). The insulin receptor is an insulin-dependent tyrosine kinase which displays autophosphorylation activity of the / subunit (Kasuga et al, 1982a,b;Van Obberghen & Kowalski, 1982;Roth & Cassell, 1983).…”