Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:l-phosphatidyl-lD-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer coIng of regulatory 85-kDa (p85) and catalytic 110-kDa (p11O) subunits. appears to be the subunit that links PI 3-kinase activity in pilO to the tyrosine-phosphorylated proteins.The insulin receptor belongs to the family of structurally related transmembrane growth factor receptors with ligandactivated protein-tyrosine kinase activity (7,8). Insulin treatment of cells has been found to increase PI 3-kinase activity in immunoprecipitates made by using antibody to phosphotyrosine (9, 10). Insulin treatment of various intact cells causes rapid tyrosine phosphorylation of a high molecular weight protein (Mr 160,000-185,000) (11, 12) termed insulin receptor substrate 1 (IRS-1) and its sequence was deduced by cDNA cloning (13). Insulin drives the formation ofa complex between tyrosine-phosphorylated IRS-1 and SH2 domains of several proteins including p85 (14-16). However, the role of the binding of PI 3-kinase to IRS-1 in insulin signal transduction is not clear. To address this issue, we disrupted complex formation between the catalytic p110 subunit of PI 3-kinase and IRS-1 by overexpressing mutant p85a (Ap85), which lacks a binding site for p110. MATERIALS AND METHODSCel Cults and Antibodies. CHO-IR cells were maintained and cultured as described (14). The antibodies used were as follows: monoclonal antibodies (mAbs) against the bovine p85a (F12 and G12) (14); polyclonal antipeptide antibodies against a synthetic C-terminal peptide of bovine p85a (residues 713-724) or bovine p85,B (residues 707-724); a polyclonal anti-pilO antibody against a glutathione S-transferase (GST) fusion protein containing residues 441-605 of bovine p110 (Transduction Laboratory, Lexington, KY); polyclonal anti-IRS-i antibodies against a synthetic rat IRS-1 peptide (pep8o) corresponding to residues 489-507 (13) or a GST fusion protein containing N-terminal residues 1-240 of rat IRS-1 (generously provided by Alan Saltiel, WarnerLambert, Ann Arbor, MI); a mAb against rat IRS-1 (ID6) (17); a mAb (py2O; ICN) and a polyclonal antibody (Upstate Biotechnology) against phosphotyrosine residues.Abbreviations: IRS-1, insulin receptor substrate 1; PtdIns, phosphatidylinositol(s); SH2, Src homology region 2; mAb, monoclonal antibody; PI(3,4,5)P3, PtdIns 3,4,5-trisphosphate; PI(4)P, PtdIns 4-phosphate; PI(4,5)P2, PtdIns 4,5-bisphosphate; PI(3)P, PtdIns 3-phosphate; PI(3,4)P2, PtdIns 3,4-bisphosphate; PI 3-kinase, 1-PtdIns 3-kinase; ATB-BMPA, 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4yloxy)-2-propylamine; PMA, phorbol 12-myristate 13-acetate.'lTo whom reprint requests should be addressed. 7415The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 sol...

Insulin, IGF‐1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3‐kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3‐kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate‐1 (IRS‐1) with PI 3‐kinase in a cell‐free system and also inhibited insulin‐ or IGF‐1‐induced, but not EGF‐induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild‐type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS‐1 with PI 3‐kinase in a cell‐free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3‐kinase activity, inhibited insulin‐ or IGF‐1‐induced membrane ruffling. These results suggest that the association of IRS‐1 with PI 3‐kinase followed by the activation of PI 3‐kinase are required for insulin‐ or IGF‐1‐induced, but not for EGF‐induced, membrane ruffling.

Insulin drives the formation of a complex between tyrosine‐phosphorylated IRS‐1 and SH2‐containing proteins. The SH2‐containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild‐type insulin receptor (IR) (CHO‐IR cells) in a cell‐free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co‐immunoprecipitated with dynamin from unstimulated CHO‐IR cells. After insulin treatment of CHO‐IR cells, anti‐dynamin antibodies co‐immunoprecipitated the IR beta‐subunit and IRS‐1, as tyrosine‐phosphorylated proteins and PI 3‐kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS‐1 or the p85 subunit of PI 3‐kinase in vitro. Together, these results suggest that in CHO‐IR cells, insulin stimulates the binding of dynamin to tyrosine‐phosphorylated IRS‐1 via Grb2 and that IRS‐1 also associates with PI 3‐kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus‐expressed IRS‐1, GST‐Grb2 fusion proteins and dynamin peptides containing proline‐rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS‐1‐derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin‐Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Both glucagon and glucagon-like peptide-1 (GLP-1) play an important role in the regulation of nutrient homeostasis. In this study, the tissue distributions of the expression of receptor genes for glucagon and GLP-1 were examined. Expression of glucagon receptor gene was detected in liver, kidney, ileum and pancreatic islets but not in brain. In contrast, expression of GLP-1 receptor gene was detected in brain, pancreas and pancreatic islets but not in liver, kidney, or ileum. To investigate the existence and characteristics of glucagon and GLP-1 receptors on pancreatic beta cells, expression of the receptor genes and translational regulation of the expression of the receptor genes by glucose were analyzed in a mouse pancreatic beta cell line, MIN6 cells. In the cDNA pool of MIN6 cells, both glucagon and GLP-1 receptor genes were identified and showed higher expression level in MIN6 cells cultured under high glucose condition than in those cultured under low glucose condition. These results suggest that glucagon and GLP-1 receptor genes are expressed in pancreatic beta cells and their expression is upregulated by glucose.

No abstract