Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor ␤-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.The insulin receptor plays important roles in metabolism and growth regulation of target tissues (1-3). Upon insulin stimulation the receptor becomes autophosphorylated on at least six or seven tyrosine residues located in the juxtamembrane, kinase, and carboxyl-terminal regions of its ␤-subunit (4 -7). Many signals are relayed through the insulin receptor substrate-1 (IRS-1), 1 which is phosphorylated by the receptor on various tyrosine residues (8). Phosphorylation of a tyrosine at position 960 in the juxtamembrane region of the insulin receptor is essential for signaling through IRS-1 (9, 10). Among the proteins that bind to IRS-1 are the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), Grb2, the tyrosine phosphatase Syp/SHPTP2, and Nck (2). IRS-1 is required for insulin-mediated mitogenesis (11, 12); however, studies using homozygous IRS-1 knockout mice predict important IRS-1-independent insulin signaling pathways (13,14). A promising candidate, the structurally closely related protein IRS-2, was recently identified, which has signaling functions similar to IRS-1 (15, 16). The Shc proteins represent additional receptor tyrosine kinase substrates that mediate p21 ras activation in the mitogenic pathway of insulin action (17). We employed the yeast two-hybrid system to identify signaling mediators for alternative pathways analogous to signals emerging from other receptor tyrosine kinases. Here we describe the identification of Grb10 as an IRS-1-independent interactive protein of the activated insulin receptor, and we define the sites of interaction in the receptor and Grb10. MATERIALS AND METHODSAntibodies-The antibody against the insulin re...

To identify receptor-associated proteins that may contribute to the specificity of insulin and IGF-I signaling responses, a mouse embryo library was screened using the yeast two-hybrid system. Multiple receptor-interactive clones encoding the SH2 domain of the adapter protein Grb10 were isolated. Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10

These data demonstrate that IL-33 serves as a potent inducer of Th2 immune response and can markedly contribute to the prolongation of cardiac allograft survival.

Protein allostery is a biological process facilitated by spatially long-range intra-protein communication, whereby ligand binding or amino acid change at a distant site affects the active site remotely. Molecular dynamics (MD) simulation provides a powerful computational approach to probe the allosteric effect. However, current MD simulations cannot reach the time scales of whole allosteric processes. The advent of deep learning made it possible to evaluate both spatially short and long-range communications for understanding allostery. For this purpose, we applied a neural relational inference model based on a graph neural network, which adopts an encoder-decoder architecture to simultaneously infer latent interactions for probing protein allosteric processes as dynamic networks of interacting residues. From the MD trajectories, this model successfully learned the long-range interactions and pathways that can mediate the allosteric communications between distant sites in the Pin1, SOD1, and MEK1 systems. Furthermore, the model can discover allostery-related interactions earlier in the MD simulation trajectories and predict relative free energy changes upon mutations more accurately than other methods.