Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

Abstract

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. Insulin binding and receptor phosphorylation were studied in a Cloudman S91 melanoma cell line and 2 of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111 showed .apprx. 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma 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. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [.gamma.-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 [sodium dodecyl sulfate] PAGE [polyacrylamide gel electrophoresis]. Insulin stimulated phosphorylation of the 95,000-MW .beta.-subunit of the receptor in all 3 cells types with similar kinetics. The amount of 32P incorporated into the .beta.-subunit in the insulin-resistant cell line 111 was .apprx. 50% of that observed with the 2 other cell lines. This difference was reflected throughout the entire dose-response curve (10-9 M to 10-6 M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the .beta.-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated 3 sites of phosphorylation in receptor from the wild type and variant 46, but only 2 major sites of phosphorylation of variant 111. The insulin-resistant variant melanoma 111 may possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.