Genetics of cell-surface receptors for bioactive polypeptides: a variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability.
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.
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004247:DNA / Q000096:biosynthesis; D003847:Deoxyglucose / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005347:Fibroblasts; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D011972:Receptor, Insulin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013696:Temperature
D007328:Insulin; D004247:DNA; D003847:Deoxyglucose; D005020:Ethyl Methanesulfonate; D011972:Receptor, Insulin