Insulin receptor signaling in Madin-Darby canine kidney cells overexpressing the human insulin receptor.

Abstract

We have developed and characterized a line of Madin-Darby canine kidney (MDCK) cells overexpressing the human insulin receptor. The expressed receptor was found to be processed normally, and its intrinsic tyrosine kinase was determined to be functional from both in vitro and in vivo phosphorylation studies. The expressed receptor was able to mediate an insulin-stimulated increase in both anti-phosphotyrosine-precipitable and anti-insulin receptor substrate 1-precipitable phosphatidylinositol 3-kinase activity. Moreover, insulin-induced glycogen synthase activity was greater and more sensitive to insulin in the transfected cells than in the parental cells. Interestingly, insulin promoted tubule-like growth in cells overexpressing the insulin receptor but not in the parental cells. Another advantage of this cell system lies in its ability to polarize into distinct basolateral and apical membrane compartments. With the use of biotinylation and Western analysis, the expressed insulin receptor was found to be preferentially expressed in the basolateral membrane (fivefold greater) in comparison with the apical membrane. Therefore, MDCK cells overexpressing the insulin receptor represent a novel system to study not only the pathway of insulin signaling, but also this pathway in the context of cell polarity.