Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations

Yip, Cecil C.; Yeung, Clement W.T.; Moule, Margaret L.

doi:10.1021/bi00542a011

Cited by 199 publications

(92 citation statements)

References 43 publications

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“…9 " 14 The successful labeling of the p and /3i subunits shown here resolves the apparent discrepancy between reports where only the a and the /3 13 or only the a and the fr 10 -11 subunits had been identified as being the components of the insulin receptor. The p and /S-, receptor subunits are structurally unrelated to the a subunit, as shown by peptide mapping experiments.…”

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confidence: 54%

Multiple Redox Forms of the Insulin Receptor in Native Liver Membranes

1980

Diabetes

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SUMMARYThree forms of disulfide-linked insulin receptor complexes are labeled by covalent cross-linking to receptor-bound 12S l-insulin in native adipocyte or liver membranes. These receptors of M r 350,000, M r 320,000, and M r 290,000 are composed of a(M r 125,000), /3(M r 90,000), and /3,(M r 49,000) subunits in stoichiometries of (a/3) 2 , {ap)(ap,), and (afi,) 2 , respectively. In adipocyte membranes, these receptor structures can undergo a first step of reduction by dithiothreitol, dissociating into M r 210,000 (a/3) and M r 160,000 (a/3,) partially reduced receptor fragments. Complete dissociation of such fragments into the free a, /3, and /S, receptor subunits is achieved at high reductant concentrations. In liver plasma membranes the partially reduced receptor species of M r 210,000 and M r 160,000 are observed even when electrophoresis is performed under nonreducing conditions. This observation indicates that native liver plasma membranes contain multiple redox states of the high affinity insulin receptor. DIABETES 29:945-947, November 1980. P revious reports from this laboratory 1 " 5 have demonstrated that insulin receptors in several tissues of the rat and human can be specifically labeled by covalent cross-linking of receptor-bound 125 l-insulin with disucciminidyl suberate and other related compounds. Dodecyl sulfate polyacrylamide gel electrophoresis, followed by autoradiographic analysis of intact rat fat cell insulin receptors labeled using this technique, showed that they exist as disulfide-linked complexes with M r 350,000." Dissociation of the M r 350,000 insulin receptor complex by dithiothreitol treatment before electrophoresis showed that it consists of 2 subunit types, the a subunit (M r 125,000) and the p subunit (M r 90.000).

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confidence: 54%

Multiple Redox Forms of the Insulin Receptor in Native Liver Membranes

1980

Diabetes

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“…One final point ofinterest from the study concerns the structure of the receptor itself (22,23). Although current models of the receptor (24) propose a heterodimer of the Mr 135,000 and 95,000 subunit linked by disulfide bonds, we also routinely observe a Mr 210,000 subunit, which has a slightly faster turnover than the other receptor subunits and is less affected by insulininduced down-regulation.…”

Section: Discussionmentioning

confidence: 91%

Insulin-induced receptor loss in cultured human lymphocytes is due to accelerated receptor degradation.

Kasuga¹,

Kahn²,

Hedo³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

212

107

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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...

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“…A coherent picture ofthe structure ofthe insulin receptor has emerged from recent studies using photoaffinity labeling (21,22), affinity crosslinking (23,24), biosynthetic labeling (25), and receptor purification (26). Specifically, the receptor appears to consist of subunits of Mr 130,000 and subunits of Mr 95,000 linked by disulfide bonds (22,25,27).…”

Section: Resultsmentioning

confidence: 99%

“…Specifically, the receptor appears to consist of subunits of Mr 130,000 and subunits of Mr 95,000 linked by disulfide bonds (22,25,27). The Mr 130,000 subunit is preferentially labeled by affinity labeling and appears to be the subunit that binds insulin; the Mr 95,000 nonbinding sub- unit is only weakly labeled after crosslinking with disuccinimidyl suberate (24,27) but is strongly labeled when the insulin receptor of IM-9 cultured lymphocytes is biosynthetically labeled with [3S]methionine and immunoprecipitated by antibodies to insulin receptors (25).…”

Section: Resultsmentioning

confidence: 99%

Structure of the insulin-like growth factor receptor in chicken embryo fibroblasts.

Kasuga¹,

Obberghen²,

Nissley³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The insulin-like growth factors (IGFs) and insulin stimulate DNA synthesis and cell multiplication in chicken embryo fibroblasts in culture. This response appears to be mediated by interaction with a single type of IGF receptor. The present study examines the subunit structure ofthis receptor by covalently crosslinking two "MI-labeled IGFs, IGF-I and multiplication-stimulating activity (MSA), to chicken embryo fibroblasts by using disuccinimidyl suberate. After solubilization, NaDodSO4polyacryl-amide gel electrophoresis, and autoradiography, IGF receptor complexes of appropriate specificity were identified; they had Mr :130,000 (major band) and -260,000 (minor band) under reducing conditions and Mr >300,000 without disulfide reduction. The proportion ofthe Mr 260,000 component increased-with increasing concentration of crosslinking agent, suggesting that it was formed from smaller proteins during the crosslinking procedure. The IGF receptor in chicken embryo fibroblasts resembles the insulin receptor in size and structure but can be. distinguished by a higher affinity for IGF-I and MSA than for insulin. Although IGF receptors with different structure and specificity have been recognized in other tissues, the function of these binding sites is unknown. The present study demonstrates that the IGF receptor of chicken embryo fibroblasts that appears to mediate the growth-promoting

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Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations

Cited by 199 publications

References 43 publications

Multiple Redox Forms of the Insulin Receptor in Native Liver Membranes

Multiple Redox Forms of the Insulin Receptor in Native Liver Membranes

Insulin-induced receptor loss in cultured human lymphocytes is due to accelerated receptor degradation.

Structure of the insulin-like growth factor receptor in chicken embryo fibroblasts.

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