The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.

The 90-kDa heat shock protein (hsp90) is a well conserved, abundant cytosolic protein believed to be a "chaperone" of most steroid receptors. We have recently demonstrated that hsp90 has an ATP-binding site and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). Circular dichroism analysis of highly purified hsp90 from rat liver shows that ATP induces an increase of beta-pleated sheet content of hsp90. Vanadate, molybdate, and heat treatment at 56 degrees C induce a similar change in the circular dichroism spectrum. Fourier transformed infrared spectroscopy reveals an ATP-induced increase in the interchain interactions of the 90-kDa heat shock protein due to an increase in its beta-pleated sheet content. In further studies we found that ATP: 1) decreases the tryptophan fluorescence of hsp90 by 11.6 +/- 1.9%; 2) increases the hydrophobic character of the protein as determined by its distribution between an aqueous phase and phenyl-Sepharose; and 3) renders hsp90 less susceptible to tryptic digestion. Our results suggest that hsp90 undergoes an "open-->closed" conformational change after the addition of ATP, analogous in many respects to the similar changes of the DnaK protein, the immunoglobulin heavy chain binding protein (BiP/GRP78), and hsp70. The ATP-induced conformational change of hsp90 may be important in regulating its association with steroid receptors and other cellular proteins.

To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 29-kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.

Insulin rapidly stimulates tyrosine phosphorylation of a 185-kDa protein in most cell types. This protein, insulin receptor substrate 1 (IRS-1), has been implicated as the first postreceptor step in insulin signal transmission based on studies with insulin receptor mutants. In cell culture and in vitro, phosphorylated IRS-1 associates with the lipid-metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), resulting in activation of this enzyme. Thus, the insulin receptor, IRS-1 and PI-3 kinase represent three of the earliest steps in insulin action at the cellular level. We have recently demonstrated that insulin is capable of stimulating PI 3-kinase activity in liver and muscle in vivo in animals and that IRS-1 phosphorylation may play a significant role in the association/activation with PI 3-kinase in vivo.

To better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3-F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half-maximal effect was observed with 1-10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane-associated insulin receptors in that they were not efficiently solubilized with 1% Triton X-100. During the same period of time, insulin stimulated nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4:Tyr1 tenfold in a time-dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS-1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with M(r) approximately 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold > 30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using 125I-BPA-insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37 degrees C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin-induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level.

Insulin and insulin-like growth factor I (IGF-I) initiate cellular functions by activating their homologous tyrosine kinase receptors. In most mammalian cell types, this results in rapid tyrosine phosphorylation of a high-molecular-weight substrate termed insulin receptor substrate 1 (IRS-1). Previous studies suggest that IRS-1 may act as a "docking" protein that noncovalently associates with certain signal-transducing molecules containing src homology 2 domains; however, direct evidence for the role of IRS-1 in the final biological actions of these hormones is still lacking. We have developed a reconstitution system to study the role of IRS-1 in insulin and IGF-I signaling, taking advantage of the fact that Xenopus oocytes possess endogenous IGF-I receptors but have little or no IRS-1, as determined by immunoblotting with anti-IRS-1 and antiphosphotyrosine antibodies. After microinjection of IRS-1 protein produced in a baculovirus expression system, tyrosyl phosphorylation of injected IRS-1 is stimulated by both insulin and IGF-I in a concentration-dependent manner, with IGF-I more potent than insulin. Furthermore, after IRS-1 injection, both hormones induce a maturation response that correlates well with the amount of injected IRS-1. By contrast, overexpression of human insulin receptors in the Xenopus oocytes does not enhance either IRS-1 phosphorylation or oocyte maturation response upon insulin stimulation. These results demonstrate that IRS-1 serves a critical role in linking IGF-I and insulin to their final cellular responses.