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Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes.

We have recently purified and cloned a new member of the insulin receptor substrate family, designated insulin receptor substrate 3 (IRS-3), from rat adipocytes. The amino acid sequence of IRS-3 shows multiple potential sites for tyrosine phosphorylation in motifs which engage specific SH2 domain-containing proteins. In order to determine which SH2 domain proteins complex with IRS-3, we have searched for coimmunoprecipitation from lysates of untreated and insulin-stimulated adipocytes. Phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP-2 complexed with the tyrosine phosphorylated form of IRS-3, whereas the phospholipase Cγ did not, and the adaptor Grb2 did so to a much lesser extent. These findings complete the survey of SH2 domain proteins associated with each of the four known members of the IRS family and provide the framework for further analysis of the role of IRS-3 in insulin signaling.

A 60-kDa protein that undergoes rapid tyrosine phosphorylation in response to insulin and then binds phosphatidylinositol 3-kinase has been previously described in adipocytes and hepatoma cells. We have isolated this protein, referred to as pp60, from rat adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA. The predicted amino acid sequence of pp60 reveals that it contains an N-terminal pleckstrin homology domain, followed by a phosphotyrosine binding domain, followed by a group of likely tyrosine phosphorylation sites, four of which are in the YXXM motif that binds to the SH2 domains of phosphatidylinositol 3-kinase. The overall architecture of pp60 is thus the same as that of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and furthermore both the pleckstrin homology and phosphotyrosine binding domains are highly homologous (about 50% identical amino acids) to these domains in both IRS-1 and IRS-2. Thus, pp60 is a new member of the IRS family, which we have designated IRS-3.

The phosphotyrosine (Tyr(P)) form of insulin receptor substrate 1 (IRS-1) is a key component in insulin signaling. Our previous study revealed that Tyr(P) IRS-1 binds to the widely distributed tyrosine phosphatase PTP2C through the src homology 2 (SH2) domains of the latter. In the present study, we examined the activity of this enzyme and of a truncated form lacking the SH2 domains (delta PTP2C) toward IRS-1 and also toward the cytoplasmic domain of the insulin receptor. Tyr(P) IRS-1 was prepared by phosphorylation of recombinant IRS-1 with recombinant cytoplasmic insulin receptor kinase (CIRK). PTP2C rapidly dephosphorylated Tyr(P) IRS-1; dephosphorylation by delta PTP2C was approximately one-third as fast. Other substrates, including Tyr(P) CIRK, were not dephosphorylated as rapidly by PTP2C; moreover, delta PTP2C was at least 10 times more active than PTP2C toward CIRK and other substrates. These results indicate that the binding of Tyr(P) residues on IRS-1 to the SH2 domain(s) of PTP2C enhances its activity toward IRS-1 and suggest that PTP2C is the phosphatase responsible for the dephosphorylation of IRS-1 in vivo. In addition, with the expectation that a PTP2C-resistant form of IRS-1 will be useful in investigations of IRS-1 function, we determined that IRS-1 can be thiophosphorylated with adenosine 5'-O-(3-thiotriphosphate) and CIRK and that this form of IRS-1 is resistant to PTP2C.

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of approximately 160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin stimulates the tyrosine phosphorylation of a 60-kDa protein (pp60) in rat adipocytes. After insulin treatment of these cells, pp60, as well as the 160-kDa insulin receptor substrate-1 (IRS-1), were found to be associated with the enzyme phosphatidylinositol 3-kinase (PtdIns-3-kinase) in separate complexes. By contrast, pp60 was not detected in insulin-treated mouse 3T3-L1 adipocytes, which contain abundant IRS-1. PtdIns-3-kinase complex. The pp60.PtdIns 3-kinase complex was located in both the soluble and membrane fractions of the rat adipocytes. Fusion proteins containing the isolated src homology 2 domains from the 85-kDa subunit of PtdIns-3-kinase bound to pp60 in lysates of insulin-treated rat adipocytes. This finding indicates that the most likely mode of association of pp60 with PtdIns-3-kinase is through binding of phosphotyrosine residues in pp60 to these domains. By immunoaffinity chromatography on a monoclonal antibody against phosphotyrosine, pp60 was purified in high percentage yield from insulin-stimulated rat adipocytes, but the low amount of the protein obtained (about 3 ng from the adipocytes of one rat) precluded sequence analysis.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.

The insulin receptor substrates (IRSs) are key proteins in signal transduction from the insulin receptor. Recently, we discovered a fourth member of this family, designated IRS-4, cloned its complementary DNA from the human embryonic kidney 293 cell line, and characterized its signaling properties in this cell line. As part of an investigation of the physiological role of this IRS, we have now cloned the mouse IRS-4 gene and determined its tissue expression and chromosomal location. The coding region of the mouse IRS-4 gene contains no introns, and in this regard is the same as that of the genes for IRS-1 and -2. The predicted amino acid sequence of mouse IRS-4 is highly homologous with that of human IRS-4; the pleckstrin homology domain, the phosphotyrosine-binding domain, and the tyrosine phosphorylation motifs are especially well conserved. The tissue distribution of IRS-4 in the mouse was determined by analysis for the expression of its messenger RNA by RT-PCR and for the protein itself by immunoprecipitation and immunoblotting. The messenger RNA was detected in skeletal muscle, brain, heart, kidney, and liver, but the protein itself was not detected in any tissue. These results indicate that IRS-4 is a very rare protein. The chromosomal locations of the mouse IRS-4 and IRS-3 genes were determined by interspecific back-cross analysis and were found to be on chromosomes X and 5, respectively. As the mouse genes for IRS-1 and -2 are on chromosomes 1 and 8, respectively, each IRS gene resides on a different chromosome.

We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.

We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné, M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211, 190-197). The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase Cgamma. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.

676 Introduction A major effect of insulin is to stimulate glucose transport into muscle and adipose tissue. This stimulation is due largely, if not entirely, to the translocation of one isotype of the glucose transporter, GLUT 4, from an intracellular vesicular pool to the plasma membrane [ 1,2]. This article reviews the current state of knowledge about insulin signalling pathways as they relate to the translocation of GLUT 4 and the stimulation of glucose transport.