2 NATURALLY-OCCURRING INSULIN-RECEPTOR TYROSINE KINASE DOMAIN MUTANTSPROVIDE EVIDENCE THAT PHOSPHOINOSITIDE 3-KINASE ACTIVATION ALONE IS NOT SUFFICIENT FOR THE MEDIATION OF INSULINS METABOLIC AND MITOGENIC EFFECTS

We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 --> Gln and Pro- 1178 --> Leu (Gln-1174 and Leu1178, respectively), both found in patie nts with inherited severe insulin resistance, markedly impaired recept or tyrosine autophosphorylation, with both mutant receptors; being una ble to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed hh Chinese hamster ovary cells, However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be suffici ent to mediate insulin's metabolic and mitogenic effects, In the prese nt study, we have demonstrated that these mutations also impair the ab ility of the insulin receptor to activate the transcription factor Elk -1 and promote GLUT4 translocation to the plasma membrane, Although at law concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at h igher insulin concentrations we confirmed that the cells expressing th e mutant receptors showed significantly increased tyrosine phosphoryla tion of IRS-1 compared with parental nontransfected cells, In addition , at comparable insulin concentrations, the association of the p85 alp ha subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and th e enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors, in contrast, no si gnificant stimulation of the tyrosine phosphorylation of Shc, GTP load ing of Ras, or mitogen-activated protein kinase phosphorylation was se en in cell lines expressing these mutant receptors. Thus, no activatio n of any measurable mitogenic or metabolic response was detectable, de spite significant insulin-induced phosphorylation of IRS-1 and its ass ociation with PI3-kinase in cells stably expressing the mutant insulin receptors, These findings suggest that PI3-kinase activation alone ma y be insufficient to mediate a wide range of the metabolic and mitogen ic effects of insulin, Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation.