Identification of insulin receptors on the insulin-independent variant 1246-3A cell line.
1246-3A cell line is an insulin-independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin-related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246-3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246-3A cells and from the parent cell line 1246 were measured; 125I-insulin binding to intact cells was 75% lower for the 1246-3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose-dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [gamma-32P]ATP. They indicated that insulin stimulated phosphorylation of the 95-kDa molecular weight beta subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin-independent cell line 1246-3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246-3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation.
D000818:Animals; D002460:Cell Line; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D007328:Insulin; D014909:Wheat Germ Agglutinins; D011972:Receptor, Insulin