Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin.
Transfected Chinese hamster ovary cell lines were developed that expressed equivalent numbers of either normal human receptor or receptor that had alanine substituted for Lys-1018 in the ATP-binding domain of the beta subunit. The mutated receptor was processed into subunits and bound insulin but lacked protein tyrosine kinase activity. Five effects of insulin were assayed: deoxyglucose uptake, S6 kinase activity, endogenous protein-tyrosine phosphorylation, glycogen synthesis, and thymidine uptake. In each case, cells bearing normal human receptors were 10-100-fold more sensitive to insulin than the parental cells. Cells with the mutant receptor behaved like the parental cells with respect to S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine uptake, but their deoxyglucose uptake was significantly depressed and relatively insensitive to insulin. The analyses led to the following conclusions: substitution of alanine for lysine at amino acid 1018 inactivates the kinase activity of the receptor; a kinase-negative receptor can be properly processed and bind insulin; insulin-dependent deoxyglucose uptake, S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine incorporation into DNA are mediated by the normal but not by the kinase-deficient human receptor.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003847:Deoxyglucose / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008239:Lysine; D008715:Methionine / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D000255:Adenosine Triphosphate; D006003:Glycogen; D003847:Deoxyglucose; D008715:Methionine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D008239:Lysine; D013936:Thymidine