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THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 266, No. 17, Issue of June 15, PP. 10995-11001, 1991 Printed in U.S.A.
Biologic Activities of Naturally Occurring Human Insulin Receptor Mutations EVIDENCETHATMETABOLICEFFECTSOFINSULIN INSULINRECEPTORMUTANT*
CAN BEMEDIATED
BY A KINASE-DEFICIENT
(Received for publication, December 21, 1990)
David E. MollerS, Heike Beneckes, and Jeffrey S. Flier From the Charles A . Dana Research Institute and Haruard-Thorndike Laboratoryof Beth Israel Hospital, Department of Medicine, Beth Israel Hospital and the Harvard Medical School, Boston, Massachusetts02215
We have studied insulin receptor-mediated signaling tantinsightsintotherelationship between structureand in Chinese hamster ovary(CHO) cell transfectants that function of the receptor. Although 17 distinct insulin receptor expressed either of two naturally occurring mutant mutations have been identified in 14 human subjects (1-12), + Ser1'O0 and Ala1134 there is as yet little human insulin receptors: TrplZo0 known about theability of natural mutants + Thr1134. Comparedwith overexpressed normal hu- to alter the normal patternof insulin-mediated signaling. In man insulin receptors, both mutant receptors displayed contrast,site-directedmutagenesis of residues withinthe normal processing and normal binding affinity;how- intracellular P-subunit has led to new insights into the conever, neither was capableof detectable insulin-stimu- sequences of impaired tyrosine kinase activity for receptor lated autophosphorylation or tyrosine kinase activity toward endogenous (ppl85) or exogenous substrates. signaling and the potential divergence of insulin-stimulated Several biologic actions of insulin were evaluated in pathways (13). Studies of the signaling properties of natural transfected cells. Compared with neomycin-only trans- mutants are also important, not only because of unique infected CHO cells (CHO-NEO), cells expressing normal sights into structure/function that may emerge, but also because it is possible that the diverse phenotypes associated receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, ['4C]glucose incorporation with severe insulin resistancemay in part be due to the ability into glycogen, [3H]thymidine incorporation into DNA, of some mutations to differentially affect insulin-regulated and specific gene expression (accumulation of glucose cellular events. transporter GLUT-1 mRNA). Cells expressing either We have recently identified two point mutations in the SerlZoo orThr1134receptors showed no increase in in- insulin receptor tyrosine kinase domain in subjects with the sulin-stimulated thymidine incorporation or GLUT- 1 Type A syndrome (14) of insulin resistance: TrpI2"'-+ Ser1200' mRNA accumulation compared with CHO-NEO. Sur- (3) and Ala1134-+ Thr":'4 (10). Only one additional naturally prisingly,cellsexpressingSerlZooreceptorsshowed occurring amino acid substitution thatis linked to an insulin increased insulin stimulation of 2-deoxyglucose uptake resistance syndrome has been identified within the intraceland glucose incorporationinto glycogen compared lular P-subunitltyrosine kinase domain (5, 16). This mutation with CHO-NEO, whereas Thr"34 receptors failed to replaced Gly"'08 within the receptor ATP-binding site and signal these metabolic responses. We conclude that 1) results in complete loss of both tyrosine kinase activity and transfected kinase-deficient insulin receptor mutants insulin-mediated biologic signaling; these effects closely rederived from insulin-resistant patients have distinct ( L ~ S ' " ~ mutations '~) defects in the ability to mediate insulin action in vitro; semble site-directedATP-bindingsite (17-19). In previous studies, the Thr113* and Ser"" mutant 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation insulin receptor cDNAs were transfected, expressed, and parof the receptor tyrosine kinase by insulin is notneces- tially characterized in Chinese hamster ovary (CHO)' cells sarily required for signaling of certain important bio- (10, 20). Inthiscontext, we showed thatinsulin-binding logic actions. affinity and receptorprocessing were normal,butinsulinstimulated autophosphorylation (in intact cells and with solubilized receptors) was severely impaired (
