Identiification of functional insulin receptors on membranes from an insulin-producing cell line (RIN5F)

Abstract

Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor α-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an M(r) 130,000 protein. The receptor β-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of M(r) 95,000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) thus display structural and functional integrity comparable with those of conventional insulin target cells.