Binding of insulin receptors to lectins: evidence for common carbohydrate determinants on several membrane receptors

Hedo, J A; Harrison, Len C.; Roth, Jesse

doi:10.1021/bi00515a013

Cited by 271 publications

(81 citation statements)

References 27 publications

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“…To characterize the equilibrium binding properties of the mutant receptors, detergent lysates of transfected cells were depleted of receptor precursor by wheat germ agglutinin affinity chromatography (29). Insulin equilibrium binding assays were then performed on the receptor isolated from the resulting glycoprotein fractions, and dissociation constants and IC 50 were determined by computerized curve fitting using a two-site sequential model as described under "Experimental Procedures."…”

Section: Resultsmentioning

confidence: 99%

“…The receptor cDNAs were expressed transiently in PEAK rapid cells using Transit 293 (Mirus) according to the manufacturer's directions. Cells were harvested by lysis in 0.15 M NaCl, 0.1 M Tris, pH 8, containing 1% Triton X-100 and protease inhibitor mixture 72 h post-transfection, and an enriched glycoprotein fraction was obtained by wheat germ agglutinin affinity chromatography (29). These fractions were stored at Ϫ80°C until assay.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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“…1% Triton X-100 (pH 7.6), bound material was eluted with 3 ml of 0.3 M N-acetylglucosamine in this buffer. Lectin chromatography allows a 20-fold purification with nearly 100% recovery of the insulin receptor (10). Immunoprecipitation of the Insulin Receptor.…”

Section: Methodsmentioning

confidence: 99%

“…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][10][11][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).…”

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confidence: 99%

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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“…After solubilization of crude membranes in 50 mM Hepes buffer (pH 7.6) with 1 % Triton X-100 and 1 mM PhMeS02F, the preparation was applied to a WGA-agarose column and receptors were eluted using 0.3 M N-acetyl-D-glucosamine in 50 mM Hepes pH 7.6 with 150 mM NaCl and 0.1 YO Triton X-100 (buffer I) [57]. 1251-IGF-I and L251-insulin binding assays were performed on the WGA-purified preparations as previously described for insulin binding assays by Harrison et al…”

Section: Igf-i and Insulin Binding Studiesmentioning

confidence: 99%

Insulin‐like growth factor I receptors on mouse neuroblastoma cells

Ota

Wilson

LeRoith

1988

European Journal of Biochemistry

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We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor j subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor /3 subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of j? subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion.Analysis of IGF-I receptor P subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.Insulin and insulin-like growth factor I (IGF-I) are polypeptides with significant structural homology, and share many common properties 11-121. IGF-I is more potent in growth promotion whereas insulin is more potent in promoting acute metabolic responses [13 -231. The growth-promoting effects are generally mediated through the IGF-I receptor, whereas metabolic effects are mediated via the insulin receptor. Recent studies have confirmed the earlier suggestions that the insulin and IGF-I receptors are distinct membrane-bound glycoproteins derived from separate genes [24-261.Specific insulin and IGF-I receptors are widely distributed on most tissues [27 -321 including nervous tissue [33 -401. We, as well as others, have demonstrated the presence of insulin and IGF-I receptors in the brain and on cells in primary culture of neuronal and glial origin [39,. Similar to insulin and IGF-I receptors on other cells, the receptors on neural-derived tissues comprise two extracellular CI subunits and two P subunits that span the membrane and contain the hormone-inducible tyrosine kinase activity in the cytoplasmic domain [44]. However, the insulin and IGF-I receptors from brain and neuronal cells are unique in having smaller apparent molecular masses, primarily because of alterations in glycosylation [36, 45 -511. In studying the insulin and IGF-I receptors on neuralderived cell lines, we recently described the presence of a phosphoprotein of 105 kDa (pp105) in cell membranes of the mouse neuroblastoma and rat glioma hybrid cell line NG108 [52]. Since this phosphoprotein could represent an endogenous substrate for the insulin and IGF-I receptors, we have attempted to characterize it further. For this purpose we have

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Binding of insulin receptors to lectins: evidence for common carbohydrate determinants on several membrane receptors

Cited by 271 publications

References 27 publications

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

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

Insulin‐like growth factor I receptors on mouse neuroblastoma cells

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