Insulin receptor structural requirements for the formation of a ternary complex with IRS-1 and PI 3-kinase.

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor tyrosine kinase and is an intermediate in insulin signaling. Phosphotyrosyl-IRS-1 binds to other signaling proteins including phosphatidylinositol 3-kinase (PI 3-kinase). We examined the role of three insulin receptor tyrosine autophosphorylation domains in association of the receptor with IRS-1. Our data support the idea that tyrosine phosphorylation of the insulin receptor juxtamembrane domain is necessary for receptor association with IRS-1. We provide evidence that the kinase regulatory domain, which is part of a loop structure at the mouth of the catalytic cleft, when mutated to replace Tyr1146, Tyr1150, and Tyr1151 with phenylalanine can bind receptor substrates without tyrosine phosphorylation of residues in the receptor juxtamembrane region. In addition, our data show that the amount of PI 3-kinase directly associated with the insulin receptor C-terminus is low when compared to the PI 3-kinase associating with IRS-1. We also demonstrate that a substantial amount (approximately 25%) of the IRS-1 associated PI 3-kinase is associated with the insulin receptor in a ternary complex of insulin receptor/IRS-1/PI 3-kinase.

Effect of phosphotyrosyl-IRS-1 level and insulin receptor tyrosine kinase activity on insulin-stimulated phosphatidylinositol 3, MAP, and S6 kinase activities.

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS-1, the insulin receptor, or both IRS-1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS-1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin-stimulated IRS-1 associated PtdIns 3'-kinase activit in these cells was 1-, 1.5-, 3-, and 3-fold, while insulin sensitivity, as determined by ED50, was 1-, 2.5-, 10-, and 10-fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl-IRS-1; however, the increased level of phosphotyrosyl-IRS-1 seen in CHO/IR/IRS-1 cells did not further increase these responses. Likewise, maximal insulin-stimulated MAP kinase activity in these cell lines increased in parallel with IRS-1 tyrosine phosphorylation except in the CHO/IR/IRS-1 cell lines with activity levels of one-, five-, nine-, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin-stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl-IRS-1, but an increase was observed with insulin-stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS-1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS-1 and other possible cellular substrates.