The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.The recent identification and cloning of proteins which interact with receptor tyrosine kinases (RTKs) has allowed much insight into the molecular basis for RTK signal transduction (9,10,18,19,24,27,54). These effector proteins contain Src homology 2 (SH2) domains of approximately 100 amino acids which interact directly with phosphotyrosine-containing regions of each RTK (21, 35). Upon receptor autophosphorylation, these SH2 domain-containing proteins interact with the RTK. Unlike most RTKs, the insulin receptor (IR) and the related insulin-like growth factor 1 receptor (IGFIR) appear to interact with and phosphorylate an intermediate signaling protein termed IRS-1, for insulin receptor substrate 1 (17). After tyrosine phosphorylation by the IR, IRS-1 is thought to interact with a variety of SH2 domain-containing proteins, including the p85 subunit of phosphatidylinositol 3-kinase, GRB-2, and SH-PTP2 (Syp) (45, 52). These proteins presumably mediate some of the effects of the IR.Another substrate of the IR is the SH2 domain-containing protein SHC, so named because of its SH2 domain as well as its homology to collagen (36). Multiple SHC proteins exist, two of which (p52 and p46) result from the use of alternative translation start sites, while the origin of the p66 isoform is less clear. The SHC proteins have been implicated in mitogenic signaling by a variety of tyrosine kinases. These include the receptors for nerve growth factor (32), epidermal growth factor (EGF) (36), platelet-derived growth factor (PDGF) (59), and insulin (39). SHC has also been implicated in signaling by other classes of receptors, including the interleukin-2 receptor (40, 62) and the T-cell receptor (41). Other hormones which have been ...

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We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/ GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an ϳ135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin-and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

The Grb10 protein appears to be an adapter protein of unknown function that has been implicated in insulin receptor (IR) signaling. The interaction of this protein with the IR has been shown to be mediated in part by the Src homology 2 (SH2) domain of Grb10. Here we demonstrate the existence of a second novel domain within Grb10 that interacts with the IR and insulin-like growth factor receptor in a kinase-dependent manner. This domain was localized to a region of approximately 50 amino acids, and we term it the BPS domain to denote its location between the PH and SH2 domains. The BPS domain does not bear any obvious resemblance to other known protein interaction domains but is highly conserved among the Grb10-related proteins Grb7 and Grb14. We show that the BPS domain interaction is dependent upon receptor tyrosine kinase activity. Furthermore, interaction of the BPS domain requires the kinase domain of the IR, since mutation of the paired tyrosine residues (Y1150F/Y1151F) within the IR activation loop dramatically reduced the interaction. Last, our data suggest that the presence of two distinct protein interaction domains may help to determine the specificity by which Grb10 interacts with different receptors. Specifically, the IR, which appears to interact most strongly with Grb10, interacts well with both the SH2 and BPS domains. Conversely, the insulin-like growth factor receptor and EGFR, which interact less avidly with Grb10, interact well only with the BPS domain or the SH2 domain, respectively. In summary, our findings demonstrate the existence of a previously unidentified tyrosine kinase activity-dependent binding domain located between the Pleckstrin homology and SH2 domains of Grb10.The binding of hormones to their receptor tyrosine kinases triggers receptor tyrosine autophosphorylation (1). In the case of the insulin receptor (IR) 1 and insulin-like growth factor I receptor (IGFIR), phosphorylation of multiple tyrosine residues leads to activation of receptor kinase activity (2) and, in addition, creates docking sites for downstream adaptor proteins such as SHC, insulin receptor substrate-1 (IRS-1), and insulin receptor substrate-2 (IRS-2) (3-5). Phosphorylation of IRS-1 upon multiple tyrosines results in its association with Src homology 2 (SH2) domain-containing proteins including Grb2, Syp, NCK, and the p85 subunit of phosphatidylinositol 3-kinase, thus activating various signaling cascades (6 -10). Tyrosyl phosphorylation of SHC also leads to its interaction with Grb2 and mediates the activation of the guanine nucleotide exchange factor SOS and subsequent activation of the Ras signaling pathway (11-14).SHC, IRS-1, and IRS-2 proteins have been shown to interact with the IR or the related IGFIR through phosphotyrosine binding domains, which recognize the phosphorylated NPXY motif in the juxtamembrane domains of receptors (3,5,15). In addition, a second poorly understood receptor binding domain has also been identified within the central domain of IRS-2 (5, 16). Both SHC and IRS-1 have been implica...

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.In recent years, extraordinarily rapid advances have been made in the understanding of the molecular mechanisms by which receptor tyrosine kinases (RTKs) function. These advances are largely due to the identification of cytosolic proteins which associate with the RTK and transduce signals from the receptor to the interior of the cell. These proteins include phospholipase C-yl, Ras-GTPase-activating protein, phosphatidylinositol 3-kinase, the SH-PTP2 phosphatase (syp), and Grb2 (11,21,22,28,29,38). All of these proteins contain Src homology 2 (SH2) domains of approximately 100 amino acids which interact directly with specific phosphotyrosine-containing peptide domains within the RTK (24, 33). The amino acid sequence surrounding the phosphotyrosine moiety (generally only four amino acids) has been shown to be the primary determinant of the specificity of SH2 domain interaction (39). From these data has emerged a general model suggesting that hormone binding induces receptor dimerization, leading to autophosphorylation on tyrosines. This results in the recruitment of SH2-containing effector proteins to the receptor (8,45). At this point, the effector protein is thought to be activated either by direct receptor phosphorylation or by other, perhaps conformational, changes which occur during this interaction. Alternatively, the receptor interaction might bring the effector protein to the plasma membrane, where it interacts with membrane-specific targets.A similar yet distinct ...