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][191][192][193][194][195][196][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 C␥. 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.The insulin receptor is a tyrosine kinase, which when activated by insulin binding phosphorylates cellular substrates. The most well characterized of these are two members of the IRS 1 family, IRS-1 and IRS-2, and the protein Shc. Tyrosine phosphorylation of the IRS proteins creates binding sites for SH2 domain-containing signaling molecules, including PI 3-kinase, the adapter molecule Grb-2, and the protein-tyrosine phosphatase SHP-2. Docking of these proteins in turn activates specific signal transduction pathways (reviewed in Refs. 1 and 2). Recently, we have identified, by purification and cloning, a third member of the IRS family, called IRS-3, which in insulintreated adipocytes is tyrosine-phosphorylated and associated with PI 3-kinase (3, 4). All three IRS family members possess a common domain structure that includes PH and PTB domains at the N terminus and, C-terminal to these, a number of potential tyrosine phosphorylation sites (1,2,4,5). The presence of these features can therefore be viewed as defining an IRS. Previously, we have identified a 160-kDa protein in HEK 293 cells, termed PY160, which is rapidly tyrosine-phosphorylated in response to insulin but which is immunologically unrelated to IRS-1 (6). In the present study we have isolated PY160 from insulin-treated HEK 293 cells and cloned its cDNA. The predicted amino acid sequence shows that PY160 is a new member of the IRS family. EXPERIMENTAL PROCEDURESCell Culture and Preparation of Lysates-HEK 293 cells were grown on 10-cm plates as described previously (6). Before use, confluent plates of cells were incubated in serum-free medium for 2 h and then incubated for 5 min further with either no addition or the addition of 1 M insulin to activate fully the insulin and IGF-1 receptors present on these cells (7). Each plate was ...

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 insulin receptor is a tyrosine kinase that is activated upon insulin binding. Signaling from this receptor proceeds primarily by its tyrosine phosphorylation of substrate proteins, which then act as docking proteins for one or more SH2 1 domain-containing proteins. Docking of these proteins in turn activates specific signal transduction pathways. The substrate docking proteins that have been molecularly characterized to date are the closely related IRS-1 and IRS-2 as well as SHC (reviewed in Refs. 1 and 2). One protein of this type that hitherto has not been cloned is a 60-kDa protein described in rat adipocytes and rat hepatoma cells. This 60-kDa protein, referred to as pp60, is rapidly tyrosine phosphorylated in response to insulin and in this form associates with the 85-kDa regulatory subunit of PI 3-kinase (3, 4). The interaction between these two proteins involves the binding of Tyr(P) to either or both of the SH2 domains of the 85-kDa subunit, because each SH2 domain by itself binds the Tyr(P) form of pp60 (3, 4). In the present study, we have isolated pp60 from insulin-treated rat adipocytes and then cloned its cDNA. The predicted amino acid sequence shows that pp60 is a new member of the IRS family. EXPERIMENTAL PROCEDURESPreparation of Affinity Matrix-GST itself and the GST fusion protein with the N-terminal SH2 domain of the 85-kDa subunit of PI 3-kinase (GST-NSH2) were prepared as described (5). These were covalently attached via sulfhydryl groups on the GST (the SH2 domain itself has no sulfhydryl groups) to activated thiopropyl Sepharose 6B beads (Sigma). The beads with coupled GST (1.3 mg/ml) or GST-NSH2 (1.8 mg/ml) were placed in columns, washed with 10 volumes of 5 mM dithiothreitol in 120 mM Tris-HCl, 1 mM EDTA, pH 6.8, to cleave the remaining 2-pyridyl disulfide, and then washed with 30 volumes of 5 mM N-ethylmaleimide in this buffer to block the free sulfhydryl groups. Protein not covalently bound to the beads was released with 25 volumes of 4% SDS, 2 mM N-ethylmaleimide, 100 mM Tris-HCl...

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a key regulator of lipid metabolism and energy balance implicated in the development of insulin resistance and obesity. The identification of putative natural and synthetic ligands and activators of PPAR-γ has helped to unravel the molecular basis of its function, including molecular details regarding ligand binding, conformational changes of the receptor, and cofactor binding, leading to the emergence of the concept of selective PPAR-γ modulators (SPPARγMs). SPPARγMs bind in distinct manners to the ligand-binding pocket of PPAR-γ, leading to alternative receptor conformations, differential cofactor recruitment/displacement, differential gene expression, and ultimately differential biological responses. Based on this concept, new and improved antidiabetic agents for the treatment of diabetes are in development. This review summarizes the current knowledge on the mechanism of action and biological effects of recently characterized SPPARγMs, including metaglidasen/halofenate, PA-082, and the angiotensin receptor antagonists, recently characterized as a new class of SPPARγMs.

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 insulintreated 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 C␥. 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.