Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: Assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates

Knutson, Victoria P.; Buck, Ruth Ann

doi:10.1016/0003-9861(91)90349-n

Cited by 3 publications

(2 citation statements)

References 13 publications

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“…Immunoblot Analysis-Samples were subjected to SDS-PAGE on discontinuous 7.5% gels, transferred to nitrocellulose, and probed with the antibodies, as described previously (40). The antibody utilized to detect the ␤ subunit of the insulin receptor and ␤Ј is denoted anti-P5 antibody, and was utilized at a concentration of 5 g/ml.…”

Section: Preparation Of Intact Insulin Receptor and Insulinmentioning

confidence: 99%

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Knutson

Donnelly

Balba

et al. 1995

Journal of Biological Chemistry

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Insulin resistance is a common clinical feature of obesity and non-insulin-dependent diabetes mellitus, and is characterized by elevated serum levels of glucose, insulin, and lipids. The mechanism by which insulin resistance is acquired is unknown. We have previously demonstrated that upon chronic treatment of fibroblasts with insulin, conditions that mimic the hyperinsulinemia associated with insulin resistance, the membrane-associated insulin receptor ␤ subunit is proteolytically cleaved, resulting in the generation of a cytosolic fragment of the ␤ subunit, ␤, and that the generation of ␤ is inhibited by the thiol protease inhibitor E64 (Knutson, V. P. (1991) J. Biol. Chem. 266, 15656 -15662). In this report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic ␤ is generated by chronic insulin administration to the cells, and that E64 inhibits the production of ␤; 2) chronic administration of insulin to the adipocytes leads to an insulinresistant state, as measured by lipogenesis and glycogen synthesis, and E64 totally prevents the generation of this insulin-induced cellular insulin resistance; 3) E64 has no effect on the insulin-induced down-regulation of insulin receptor substrate-1, and therefore insulin resistance is not mediated by the down-regulation of insulin receptor substrate-1; 4) under in vitro conditions, partially purified ␤ stoichiometrically inhibits the insulin-induced autophosphorylation of the insulin receptor ␤ subunit; and 5) administration of E64 to obese Zucker fatty rats improves the insulin resistance of the rats compared to saline-treated animals. These data indicate that ␤ is a mediator of insulin resistance, and the mechanism of action of ␤ is the inhibition of the insulin-induced autophosphorylation of the ␤ subunit of the insulin receptor.Insulin resistance is a characteristic clinical feature of a number of disease states, chief among them diabetes mellitus, and is associated with hyperglycemia, hyperinsulinemia, hyperlipidemia, and hypertension (reviewed in Refs. 1 and 2). The potential mechanisms by which cellular insulin resistance is generated are many: a mutation in the gene coding for the insulin receptor protein, resulting in a decreased expression of the protein; a decrease in the binding of insulin; a decrease in the number of insulin receptor molecules expressed on the plasma membrane of the target cell; or a so-called "post-receptor defect" in which there is a decreased interaction between the insulin receptor and downstream effector molecules.Natural mutations in the primary sequence of the insulin receptor have been identified in patients with extreme forms of insulin resistance (3, 4). Mutations in the extracellular ligand binding domain of the receptor have been shown to result in a decreased affinity of insulin for the receptor (5-9). Mutations have also been documented in the intracellular ␤ subunit of the receptor, especially in the ATP binding domain and the autophosphorylation domain of the receptor protein, resulting in a decreased tyrosine kinase activity...

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Section: Preparation Of Intact Insulin Receptor and Insulinmentioning

confidence: 99%

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Knutson

Donnelly

Balba

et al. 1995

Journal of Biological Chemistry

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“…The molecular mass of the BAE cell insulin receptor p subunit was also determined. Immuno-blot analysis of the p subunit with an anti-peptide antibody specific for the receptor p subunit (Knutson and Buck, 1991) demonstrated a mass of 109 kDa (data not shown). The mass characteristically observed for the p subunit is 92-95 kDa (Zick, 1989).…”

Section: Discussionmentioning

confidence: 97%

Insulin receptor characterization and function in bovine aorta endothelial cells: Insulin degradation by a plasma membrane, protease‐resistant insulin receptor

Milton¹,

Knutson²

1993

Journal Cellular Physiology

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The functional significance of the insulin receptor on bovine aorta endothelial (BAE) cells is not well defined. The insulin receptor expressed on BAE cells does not mediate insulin hormonal effects and does not mediate the transcytosis of insulin from the apical to the basolateral domain of the cell monolayer. To assess the role of the insulin receptor on BAE cells, the physical characteristics of the BAE cell receptor were investigated, and the time-dependent interaction of insulin and insulin degradation products with BAE cell monolayers was quantitated. The BAE cell insulin receptor was found to be highly resistant to the proteolytic action of trypsin, pronase, and proteinase K at either 4 degrees C or 37 degrees C. This resistance may permit the receptor to maintain insulin binding capabilities in spite of the high concentrations of proteases which are normally present in blood. Scatchard analysis of cell-surface and total cellular insulin receptor demonstrated dissociation constants similar to values obtained with other cells and tissues. However, whereas other cells and tissues contain an intracellular pool of receptor that ranges from 20-40% of the total cellular receptor content, no intracellular population of insulin receptors was detected in BAE cells. Upon incubation of intact BAE cell monolayers with insulin, no endocytosis of cell-surface insulin receptor could be demonstrated. However, insulin degradation by the BAE cells was readily quantitated, at a rate of 16.3 fmol/10(6) cells/h at an insulin concentration of 2 nM. This rate of degradation was not inhibited by chloroquine, which inhibits insulin degradation in fibroblasts, hepatocytes, and adipocytes, nor by phenylarsine oxide, which inhibits endocytosis. Bacitracin inhibited insulin binding to the cell monolayers and inhibited insulin degradation with identical IC50 values (80 microM). These data suggest that in BAE cells, insulin degradation occurs in the absence of receptor-mediated endocytosis and is mediated by binding of insulin to its receptor. Therefore, it is concluded that the functional role of the insulin receptor expressed in BAE cells is to bind blood-borne insulin at the plasma membrane of the cell and thereby facilitate the degradation of insulin at the BAE cell plasma membrane.

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Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

Knutson¹

1991

Journal of Biological Chemistry

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Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: Assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates

Cited by 3 publications

References 13 publications

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Insulin receptor characterization and function in bovine aorta endothelial cells: Insulin degradation by a plasma membrane, protease‐resistant insulin receptor

Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

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