Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

Abstract

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 ...

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

“…A number of docking molecules contain both a PH domain and a PTB domain, which essentially provides two contributions to membrane localization for the host protein. In the case of IRS-1, intact PH and PTB domains are required for e cient coupling of IRS-1 to the insulin receptor and tyrosine phosphorylation of IRS-1 (O'Neill et al, 1994;Gustafson et al, 1995;Yenush et al, 1996). The role of the PH domains of Dok and Dok-R is currently unknown, although they can be expected to perform a similar function in mediating phospholipid binding and subsequent membrane targeting upon receptor stimulation.…”

“…As an approach to identify molecules that are involved in Tek signaling pathways, we have used the yeast twohybrid system to ®nd proteins that can interact with the intracellular domain of Tek in a phosphotyrosinedependent manner (Fields and Song, 1989;Chien et al, 1991;O'Neill et al, 1994;Pandey et al, 1994). The entire intracellular domain of the murine receptor (amino acids 780 ± 1122) (Tek IC ) was fused in-frame to the DNA binding domain of the Escherichia coli (E. coli) LexA transcriptional activator and this resulted in constitutive tyrosine phosphorylation of the fusion protein ( Figure 1a).…”

“…Functional deletion analysis of the Dok-R cDNA demonstrated that a small region encompassing amino acids 141 ± 271 is su cient for binding to Tek IC in yeast (Figure 1d). This region shows sequence conservation with the minimal domain required for phosphotyrosine binding in the insulin receptor substrate-1 (IRS-1) (O'Neill et al, 1994;Gustafson et al, 1995) and it also possesses the critical arginine residues thought to contact phosphotyrosine in the IRS-family PTB domains (Eck et al, 1996;Zhou et al, 1996) (Figure 1e). The phosphotyrosine binding (PTB) domains of Shc and IRS-1 have been shown to bind to NPXY motifs in target proteins (Kavanaugh et al, 1995;Wolf et al, 1995;Gustafson et al, 1995); however, the Tek receptor does not contain such a sequence motif suggesting that the PTB domain of Dok-R may recognize a di erent target amino acid sequence.…”

Re-engineering the target specificity of the insulin receptor by modification of a PTB domain binding site

“…The phosphotyrosine (pTyr)-binding (PTB) domain (also referred to as the PI domain) was originally identi®ed in the Shc and IRS-1 docking proteins, through its ability to recognize speci®c pTyr-containing sites on activated receptor protein tyrosine kinases (Blaikie et al, 1994;Kavanaugh and Williams, 1994;O'Neill et al, 1994;van der Geer et al, 1995). These PTB domains are functionally similar to SH2 domains in the sense that they bind directly to short peptide motifs in a fashion that is regulated by phosphorylation of the ligand, and depends on residues¯anking the phosphorylation site (Songyang et al, 1995).…”

The SH2-containing adapter protein GRB10 interacts with BCR-ABL

“…To screen for phosphotyrosine-dependent protein ± protein interactions, we performed a modi®ed yeast two-hybrid screen with Bcr-Abl. Autophosphorylation of Bcr-Abl in this modi®ed yeast system was accomplished by using the DNA-binding protein LexA that leads to dimerization and crossphosphorylation of the fused bait (O'Neill et al, 1994;Weidner et al, 1996). In this article, we describe the identi®cation of Grb10, a novel SH2-containing protein, which interacts with Bcr-Abl in yeast, in vitro and in vivo.…”