Genetics of cell-surface receptors for bioactive polypeptides: a variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability

Abstract

An insulin-nonresponsive variant was isolated from mutagenized mouse BALBc/3T3 fibroblasts. Selection was based on the insulin's mitogenic action upon quiescent cells and subsequent arrest at mitosis by vinblastine sulfate to remove insulin-responsive cells. Among four surviving colonies, one, designated IN-2, exhibited no binding for [123I] insulin at 2 x 10(-10) M and at 4 degrees C. The binding ability, however, recovered substantially at 15 degrees C and increased with higher temperature and at higher ligand concentrations. The binding profiles, Scatchard plot analysis, and the dissociation kinetics indicated that the receptors expressed on IN-2 cells possess lower affinity than the parental 3T3 cells. The IN-2 cells were negative for stimulating effects of insulin on 2-deoxyglucose uptake, thymidine incorporation, and cell growth. The IN-2 cells were also negative for cross-reactivity to antibodies which react with insulin receptors on 3T3 cells and for the susceptibility to a cytotoxic chimeric insulin which was cross-linked to diphtheria toxin fragment A. This negative response of IN-2 cells can be attributed to a deficiency in "high-affinity receptors" for insulin. The insulin bound to the "low-affinity binding sites" of IN-2 cells, however, undergoes internalization and intracellular degradation. Therefore, such processing by itself does not account for insulin's mitogenic action.