Insulin receptor: covalent labeling and identification of subunits.

Jacobs, Steven; Hazum, Eli; Shechter, Yoram; Cuatrecasas, Pedro

doi:10.1073/pnas.76.10.4918

Cited by 238 publications

(106 citation statements)

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“…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.…”

Section: Vol 235mentioning

confidence: 67%

“…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). This suggests that the insulin binding site is located on the a-subunit of the receptor oligomer.…”

Section: Vol 235mentioning

confidence: 99%

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Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

138

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The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Vol 235mentioning

confidence: 67%

Section: Vol 235mentioning

confidence: 99%

Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

138

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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: 93%

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.

211

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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“…In temis of size and reducibility, the bombyxin receptor reveals similarities to the insulin receptor, which is a heterotetramer of an apparent molecular mass of 310 kDa [26,271. After reduction, the insulin receptor forms two insulin-binding a-subunits (molecular mass 135 kDa) and two transmembrane P-subunits (molecular mass 90 kDa).…”

Section: Resultsmentioning

confidence: 99%

The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells

Fullbright

Lacy

Büllesbach

1997

European Journal of Biochemistry

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Bombyxin 11, a product of the brain of the adult silkmoth, Bombyx mori, binds to ovarian cells of three different species of lepidoptera, i.e. B. mori (silkmoth), Samia Cynthia ricini (ailanthus moth), and an ovarian cell line of Spodopteru frugiperda (Sf9) (fall armyworm). Crude Sf9 cell membrane preparations were used to show that the purported bombyxin receptor binds its ligand in a specific, saturable, and reversible manner. The dissociation constant of the bombyxin-receptor complex is 260 -C 90 pM. Quantitative binding studies and Scatchard analysis suggest that every Sf9 cell displays 20000 receptors on the surface. The cross-linked bombyxin-receptor ligand complex has an apparent molecular mass of about 300 kDa as determined by SDSIPAGE. Reduction causes the bombyxin receptor to dissociate into two subunits with molecular masses of 90 kDa and 116 kDa. The size and subunit structure of the putative bombyxin receptor on Sf9 cells show some similarities to the mammalian insulin receptor.Keywords: bombyxin ; insulin ; bombyxin receptor; structure/function relationship ; photoactivatible crosslink Bombyxin, an invertebrate developmental factor similar in structure to insulin, was isolated from the brain of the silkmoth, Bombyx mori [I, 21. Bombyxin elicits metamorphosis in brain extirpated dormant pupae of the saturniid moth, Sumia Cynthia ricini [3], by acting on the prothoracic gland to stimulate the synthesis and release of ecdysone, the steroid hormone that causes molting and metamorphosis. This effect is ascribed to two structurally unrelated prothoracicotropic hormones, the smaller of which was later named bombyxin, while the larger one (30 kDa) retained the name prothoracicotropic hormone [4, 51. The structural dissimilarity of the two hormones means that there are different receptors that mediate a similar cell response. Indeed, the prothoracicotropic glands of Manduca sexta were sensitive to the small and large prothoracicotropic hormones, as demonstrated by Bollenbacher et al. [6].Although initial observations pointed to bombyxin as a prothoracicotropic hormone 171, its biological function in B. mori remained obscure [8, 91. Bombyxin titers in the hemolymph of postembryonic B. mori showed no correlation with ecdysone during molting [ 101. Furthermore, applications of physiological concentrations of bombyxin to brain extirpated dormant B. mori were ineffective in inducing metamorphosis [3].Based on the hypothesis that a structural relationship may indicate functional similarity, the insulin-like bombyxin has been suspected to have an effect on the blood sugar of insects, for instance in the metabolism of trehalose [Ill. Bombyxin was detected in the ovaries of the silkmoth [I21 and significantly higher levels of bombyxin were found in the hemolymph of the female [lo]. This observation suggests a role for bombyxin similar to that of the mammalian ovarian hormone relaxin, another member of the insulin family. It seems possible that bombyxin is involved in the ovarian development [13] of several specie...

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Insulin receptor: covalent labeling and identification of subunits.

Cited by 238 publications

References 23 publications

Protein kinase activity of the insulin receptor

Protein kinase activity of the insulin receptor

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

The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells

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