title: Insulin receptor characterization and function in bovine aorta endothelial cells: insulin degradation by a plasma membrane, protease-resistant insulin receptor.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Texas Medical School at Houston 77225.
authors.forename: S G
authors.initials: SG
authors.lastname: Milton
authors.forename: V P
authors.initials: VP
authors.lastname: Knutson
mesh_terms: D000818:Animals; D001011:Aorta; D001414:Bacitracin / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004351:Drug Resistance; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D019286:Endopeptidase K; D010450:Endopeptidases / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D011402:Pronase / Q000494:pharmacology; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism / Q000502:physiology*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D007328:Insulin; D007457:Iodine Radioisotopes; D001414:Bacitracin; D002738:Chloroquine; D011972:Receptor, Insulin; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D011402:Pronase