Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins.

Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use.

In recent years, analogs of human insulin have been engineered with the aim of improving therapy for people with diabetes. To ensure that the safety profile of the human hormone is not compromised by the molecular modifications, the toxico-pharmacological properties of insulin analogs should be carefully monitored. In this study, we compared the insulin and IGF-I receptor binding properties and metabolic and mitogenic potencies of insulin aspart (B28Asp human insulin), insulin lispro (B28Lys,B29Pro human insulin), insulin glargine (A21Gly,B31Arg,B32Arg human insulin), insulin detemir (NN304) [B29Lys(epsilon-tetradecanoyl), desB30 human insulin], and reference insulin analogs. Receptor affinities were measured using purified human receptors, insulin receptor dissociation rates were determined using Chinese hamster ovary cells overexpressing the human insulin receptor, metabolic potencies were evaluated using primary mouse adipocytes, and mitogenic potencies were determined in human osteosarcoma cells. Metabolic potencies correlated well with insulin receptor affinities. Mitogenic potencies in general correlated better with IGF-I receptor affinities than with insulin receptor off-rates. The 2 rapid-acting insulin analogs aspart and lispro resembled human insulin on all parameters, except for a slightly elevated IGF-I receptor affinity of lispro. In contrast, the 2 long-acting insulin analogs, glargine and detemir, differed significantly from human insulin. The combination of the B31B32diArg and A21Gly substitutions provided insulin glargine with a 6- to 8-fold increased IGF-I receptor affinity and mitogenic potency compared with human insulin. The attachment of a fatty acid chain to LysB29 provided insulin detemir with reduced receptor affinities and metabolic and mitogenic potencies but did not change the balance between mitogenic and metabolic potencies. The safety implications of the increased growth-stimulating potential of insulin glargine are unclear. The reduced in vitro potency of insulin detemir might explain why this analog is not as effective on a molar basis as human insulin in humans.

The metabolisable hexoses D-glucose and D-mannose enhance the expression of IRS-2 but not of IRS-1 in pancreatic beta-cells.

D-glucose regulates maintenance and function of pancreatic beta-cells. Several studies have shown that IRS-2, but not IRS-1, is necessary to maintain and sufficient to expand functional beta-cell mass. We therefore analyzed the expression of IRS-2 and IRS-1 in beta-cells after culture in the presence of various concentrations of D-glucose and other metabolisable or non-metabolisable hexoses. D-glucose increased Irs-2 transcription and IRS-2 accumulation in a dose-dependent manner (1.6 to 25 mmol/l), with a 3-fold increased plateau after 10 h. In contrast, the expression of IRS-1 remained unaffected. D-glucose also induced phosphorylation of IRS-2 while non-metabolisable hexoses did neither affect expression nor phosphorylation. D-glucose-mediated elevation and phosphorylation of IRS-2 were independent of autocrine insulin action although insulin itself could transiently and slightly enhance IRS-2 expression.

Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results.

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.

Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human beta-cells from hyperglycemia-induced apoptosis.

Studies in vivo indicate that IRS2 plays an important role in maintaining functional beta-cell mass. To investigate if IRS2 autonomously affects beta-cells, we have studied proliferation, apoptosis, and beta-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that beta-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a beta-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of beta-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human beta-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve beta-cell function. Our results indicate that IRS2 acts autonomously in beta-cells in maintenance and expansion of functional beta-cell mass in vivo.

T1 is a c-Fos- and FosB-responsive gene which is induced by growth factors through multiple signal transduction pathways.

Stimulation of quiescent cells with growth factors triggers changes in gene expression through multiple signal transduction pathways. One of these changes in Swiss 3T3 cells is the strong accumulation of T1 mRNA which encodes a secreted glycoprotein of the immunoglobulin superfamily. Proliferating cells continued to express T1 mRNA at a lower level, whereas growth arrest induced either by serum deprivation or by contact inhibition was paralleled by the disappearance of the T1 mRNA. T1 mRNA synthesis in response to serum and platelet-derived growth factor stimulation is mediated through protein kinase C-dependent and protein kinase C-independent pathways. Activation of protein kinase A also led to T1 gene expression. Ongoing protein synthesis is a prerequisite for T1 gene induction by growth factors which defines T1 as a delayed early serum-responsive gene. The ability of the immediate early transcription factors c-Fos and FosB to directly induce the T1 gene was demonstrated in a conditional expression system in the absence of protein synthesis. Furthermore, all known inducers of the T1 gene also lead to c-fos gene activation. Thus we show that the T1 gene is regulated by signals which are transduced through multiple pathways and provide evidence that the Fos proteins play an important role in the integration of these pathways.

Growth factor-mediated induction of the delayed early gene T1 depends on a 12-O-tetradecanoylphorbol 13-acetate-responsive element located 3.6 kb upstream of the transcription initiation site.

The T1 gene is a delayed early serum-responsive gene which encodes a secreted glycoprotein of the immunoglobulin superfamily. We have addressed the question of what promoter elements are needed to allow for growth factor-mediated T1 gene expression. By deletion analysis we have identified a 448-bp DNA region 3.5-4.0 kb upstream of the transcription start site which can confer serum inducibility onto a foreign minimal promoter. Within this sequence there is a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE) which is essential for T1 promoter induction in response to the forced expression of the transcription factor AP-1 in NIH 3T3 fibroblasts and F9 teratocarcinoma cells. This TRE is crucial for growth factor-mediated T1 gene expression. A point mutation within this TRE attenuated serum inducibility. Two E boxes are positioned 6 and 40 bp downstream of the TRE. Point mutations within these sequence motifs reduced basal T1 promoter activity and serum inducibility. Additional, as-yet-unidentified, promoter elements within the 448-bp serum-responsive region are required for T1 gene activation in response to growth stimulation.

Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2.

The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions. The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain. SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors. As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis. Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides. A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A. The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction.

Cooperative interactions of AP-1 and basic helix-loop-helix transcription factors regulate T1 gene expression.

The T1 gene is a murine, delayed early serum-responsive gene that encodes glycoproteins of the interleukin-1 receptor (IL-1R) family. Transcription of the T1 gene leads to production of two mRNAs that encode a transmembrane protein, which is highly similar to the type-1 IL-1R, and a secreted protein, which consists solely of the extracellular part. Fibroblasts, in contrast to mast cells, express predominantly the shorter form of the protein, and several mitogens cause strong, transient induction of the T1 gene in these cells. Here we describe the identification of a 148 bp enhancer element that is positioned 3.6 kb upstream of the transcription initiation site. A TPA-responsive element (TRE) and three identical E-boxes are located within this sequence. Introduced point mutations confirmed the necessity of these sites for full T1 promoter activity. The TRE and the distal E-box are absolutely indispensable for promoter function, whereas the two proximal E-boxes contribute less to promoter strength. In vitro the three E-boxes are bound by different protein complexes.

The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling.

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.

Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor.

SUMMARY: Crystal structures of the insulin receptor substrate-1 (IRS-1) phosphotyrosine-binding (PTB) domain, alone and complexed with the juxtamembrane region of the insulin receptor, show how this domain recognizes phosphorylated "NPXY" sequence motifs. The domain is a 7-stranded beta sandwich capped by a C-terminal helix. The insulin receptor phosphopeptide fills an L-shaped cleft on the domain. The N-terminal residues of the bound peptide form an additional strand in the beta sandwich, stabilized by contacts with the C-terminal helix. These interactions explain why IRS-1 binds to the insulin receptor but not to NPXpY motifs in growth factor receptors. The PTB domains of IRS-1 and Shc share a common fold with pleckstrin homology domains. Overall, ligand binding by IRS-1 and Shc PTB domains is similar, but residues critical for phosphotyrosine recognition are not conserved.

Specificity of the PTB domain of Shc for beta turn-forming pentapeptide motifs amino-terminal to phosphotyrosine.

Shc phosphorylation in cells following growth factor, insulin, cytokine, and lymphocyte receptor activation leads to its association with Grb2 and activation of Ras. In addition to being a cytoplasmic substrate of tyrosine kinases, Shc contains an SH2 domain and a non-SH2 phosphotyrosine binding (PTB) domain. Here we show that the Shc PTB domain, but not the SH2 domain, binds with high affinity (ID50 approximately equal to 1 microM) to phosphopeptides corresponding to the sequence surrounding Tyr250 of the polyoma virus middle T (mT) antigen (LLSNPTpYSVMRSK). Truncation studies show that five residues amino-terminal to tyrosine are required for high affinity binding, whereas all residues carboxyl-terminal to tyrosine can be deleted without loss of affinity. Substitution studies show that tyrosine phosphorylation is required and residues at -5, -3, -2, and -1 positions relative to pTyr are important for this interaction. 1H NMR studies demonstrate that the phosphorylated mT antigen-derived sequence forms a stable beta turn in solution, and correlations between structure and function indicate that the beta turn is important for PTB domain recognition. These results show that PTB domains are functionally distinct from SH2 domains. Whereas SH2 domain binding specificity derives from peptide sequences carboxyl-terminal to phosphotyrosine, the Shc PTB domain gains specificity by interacting with beta turn-forming sequences amino-terminal to phosphotyrosine.

Insulin aspart: a novel rapid-acting human insulin analogue.

In order to improve therapy and increase the quality of life for diabetic patients, it has been of significant interest to develop rapid-acting insulin preparations that mimic the physiological meal-time profile of insulin more closely than soluble human insulin. Insulin aspart (B28Asp human insulin) is a novel rapid-acting insulin analogue that fulfils this criterion. The B28Asp modification weakens the self-association of the insulin molecule and provides a more rapid absorption from the sc. injection site. The preclinical evaluation in vitro and in vivo demonstrates that apart from the more rapid absorption, insulin aspart is equivalent to human insulin. Thus, insulin aspart is equivalent to human insulin on key in vitro parameters such as insulin receptor affinity, insulin receptor dissociation rate, insulin receptor tyrosine kinase activation, IGF-I receptor binding affinity, metabolic and mitogenic potency. In accordance with the equivalent in vitro profiles, the toxico-pharmacological properties of insulin aspart and human insulin are also identical. The available data for insulin aspart and other rapid-acting insulin analogues supports that in vitro assays are sensitive and valuable in the preclinical evaluation of insulin analogues. Clinical studies demonstrate that insulin aspart has a pharmacokinetic and pharmacodynamic profile superior to that of soluble human insulin. In Type 1 diabetic patients on a basal-bolus injection regimen, insulin aspart given immediately before the meals provides an improved postprandial glycaemic control and an improved long-term metabolic control, as compared to soluble human insulin given 30 min before the meals, without increasing the risk of hypoglycaemia. Taken together, the data support the hope that insulin aspart will allow the diabetic patient to combine a more flexible lifestyle with better glycaemic control, without any increased safety risk.

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38.

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase.

Previously, we have identified p120 as a Fyn/Lck SH3 and SH2 domain-binding protein that is tyrosine phosphorylated rapidly after T cell receptor triggering. Here, we used direct protein purification, amino acid sequence analysis, reactivity with antibodies, and two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes of p120cbl with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120cbl with Fyn and the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was markedly increased by T cell activation, consistent with in vitro binding of p120cbl to their SH2 as well as SH3 domains. In contrast, a large fraction of p120cbl was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120cbl interacted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120cbl signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120cbl with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.

PTB domains of IRS-1 and Shc have distinct but overlapping binding specificities.

PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding domains originally described in the receptor tyrosine kinase substrate, Shc. By serial truncation, we show that a 174-residue region of Shc p52 (33-206) has full PTB activity. We also show that a 173-residue region of insulin receptor substrate-1 (IRS-1; residues 144-316) has related PTB activity. In vitro both domains bind directly to activated insulin receptors. Binding is abrogated by substitution of Tyr-960 and selectively inhibited by phosphopeptides containing NPXY sequences. Phosphopeptide assays developed to compare PTB domain specificities show that the Shc PTB domain binds with highest affinity to psi XN beta 1 beta 2 pY motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth factor receptors (psi = hydrophobic, beta = beta-turn forming); the IRS-1 PTB domain does not bind with this motif. In contrast, both the Shc and IRS-1 PTB domains bind psi psi psi XXN beta 1 beta 2pY sequences derived from insulin and interleukin 4 receptors, although specificities vary in detail. Shc and IRS-1 are phosphorylated by distinct but overlapping sets of receptor-linked tyrosine kinases. These differences may be accounted for by the inherent specificities of their respective PTB domains.

Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain.

We present the NMR structure of the PTB domain of insulin receptor substrate-1 (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Despite the lack of sequence homology and different binding specificity, the overall fold of the protein is similar to that of the Shc PTB domain and closely resembles that of PH domains. However, the PTB domain of IRS-1 is smaller than that of Shc (110 versus 170 residues) and binds to phosphopeptides in a distinct manner. We explain the phosphopeptide binding specificity based on the structure of the complex and results of site-directed mutagenesis experiments.