We have measured the turnover rate ofthe polypeptide subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) and have investigated the mechanism of insulin-induced receptor loss. To estimate the rate of receptor degradation, lymphocytes were either pulse-labeled with [asSimethionine or surface labeled with NaF4 and lactoperoxidase. The insulin receptor was isolated by immunoprecipitation with anti-receptor antibody, and the rate of loss of radioactivity from each receptor subunit was determined after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major (Mr 135,000 and 95,000) and one minor (Mr 210,000) subunits were found. By both labeling methods, the half-lives of the major insulin receptor subunits were 9-12 hr in normal media. When the cells were cultured in media containing 1 FAM insulin the turnover was accelerated 2.5-to 3.5-fold (half-life approximately 3 hr). The increase in degradation rate was dependent on the insulin concentration and correlated well with the ability to "down-regulate" the receptor. Guinea pig insulin was about 2% as active as porcine insulin in accelerating degradation, and human growth hormone was without effect. The acceleration of receptor degradation induced by insulin was partially blocked by 100 pM cycloheximide. The rate of biosynthesis of the insulin receptor did not appear to be altered in the presence of 1 jpM insulin after correction for the change in degradation rate. In conclusion, these data demonstrate that insulin-induced receptor loss in cultured lymphocytes is due to accelerated receptor degradation. The ability of hormones to regulate the concentration of their receptors on the surface of cells ("down-regulation") is a fundamental mechanism for the regulation of target cell sensitivity (1). Lymphocytes cultured in media containing various concentrations of insulin exhibit a time-and concentration-dependent decrease in insulin receptor concentration (2). Similarly, the number of insulin receptors on cells in vivo in many diseases is inversely related to the concentration ofinsulin to which the cells are exposed (3). This mechanism of insulin-induced loss of its own receptor is thought to play a major role in the pathogenesis of insulin resistance in many disease states, including obesity (3,4).Using autoantibodies against the insulin receptor, we have recently identified the receptor subunits by specific immunoprecipitation of either externally or biosynthetically labeled proteins (5-7). In the present study we have used these techniques to measure directly the turnover rate ofinsulin receptor subunits and to study the mechanism of down-regulation of insulin receptors in human cultured lymphocytes. MATERIALS AND METHODSMaterials. Porcine insulin was purchased from Elanco (Indianapolis, IN); guinea pig insulin and human growth hormone were gifts from the Research Resources Program, and the National Pituitary Agency, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health.Na'"I, [a...
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.
[3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. NaDodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component Of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.Most, if not all, cell surface proteins and receptors are believed to contain covalently attached carbohydrate side chains-i.e., they are glycoproteins. Along these lines, the insulin receptor, an integral membrane protein (1), is also believed to be glycosylated. This suggestion was first made based on studies that showed impairment ofinsulin binding after digestion offat cells with glycosidases (2) and on the observation that concanavalin A and wheat germ agglutinin alter insulin binding and exhibit insulin-like effects (3). Furthermore, complexes of both lectins linked to agarose retain the solubilized insulin receptor (3). Recently, the interaction ofthe solubilized insulin receptor with a panel of 12 different lectins was studied and, on this basis, it was suggested that the carbohydrate moiety contains mainly Nacetylglucosamine, galactose, and mannose (4).To characterize directly the glycosylation of the insulin receptor, we have biosynthetically labeled the receptor of cultured human lymphocytes (IM-9 line) with tritiated monosaccharides or externally labeled the receptor by using procedures that preferentially react with galactose and sialic acid residues (5-8). To detect the insulin receptor, we used immunoprecipitation with sera from patients who have type B insulin resistance and acanthosis nigricans that contain autoantibodies that react specifically with the receptor for insulin (9-11). These studies not only directly reveal the glycoprotein nature of both ma...
[125I]Insulin binding has been studied in two patients with extreme insulin resistance using cultured B-lymphocytes transformed with Epstein-Barr virus. A cell line from a female infant with leprechaunism had insulin binding which was decreased 90% below the lower limit of normal. Lymphocytes from a young woman with type A extreme insulin resistance (associated with acanthosis nigricans and virilization) had insulin binding which was 80% depressed. In both cases, the defect in binding resulted from a decrease in the number of receptors per cell. The remaining receptors had normal properties, including a normal affinity for insulin and a normal specificity for insulin analogs. Insulin binding in cultured lymphocytes from these two insulin-resistant patients was also inhibited normally by antibodies to the insulin receptor. Immunological assays using anti-receptor antibodies confirmed the conclusion that the number of receptors was decreased. Affinity labeling of the leprechaun insulin receptor with [125I]insulin demonstrated the existence of an alpha-subunit with apparently normal molecular weight (130,000 daltons). However, the number of receptor molecules per cell appeared reduced.
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