(Scanned from the applicant's description) The overall goals of this research proposal are to identify and characterize in the insulin signal transduction pathways leading to the functional regulation of the protein components directly involved in the intracellular trafficking of GLUT4-containing vesicles. Recently, we have determined that a novel small GTP binding protein in the Rho family (TC1O) provides essential function for the insulin-stimulated translocation of GLUT4 in adipocytes. The functional properties of TC1O are dependent upon several factors including appropriate intracellular compartmentation and insulin-dependent activation into the GTP-bound state. This is specific for TC1O as other Rho family members, RhoA, RhoD and CDC42) are without effect. In parallel, we have also observed that CAP plays an important role in the regulation of TC10 which apparently occurs through its ability to recruit tyrosine phosphorylated Cbl to the lipid raft enriched plasma membrane subdomains. In turn, the tyrosine phosphorylated Cbl protein can engage the small adapter protein CrkIl through its SH2 domain. Furthermore, the central SH3 domain of Crkll directly associates with C3G which functions as a guanylnucleotide exchange factor (GEF) for TC1O. Importantly, the assembly of this signaling cascade occurs in the lipid raft plasma membrane subdomain due to the specific binding of CAP to the caveolar protein flotillin. In addition, the insulin-stimulated recruitment of the CAP/Cbl complex and the activation of TC10 through the engagement of the Crkll/C3G complex occur independent of and parallel to the signaling pathway mediating P1 3-kinase activation. Thus, the specific aims of this proposal are directed at defining the mechanisms by which insulin utilizes a novel compartmentalized signal transduction pathway that functions in concert with the P1 3-kinase pathway to stimulate GLUT4 translocation. To accomplish these objectives we will utilize Cbl knock-out mice to examine the role of CAP/Cbl recruitment in the activation of TC10 and its coupling to GLUT4 translocation. We will examine the molecular mechanism by which C3G can specifically active TC10 and then identify potential GAPs and downstream effectors directly leading to the regulation of GLUT4 translocation. Subsequently, we will assess the role of plasma membrane lipid raft subdomain targeting as a requirement for appropriate signal generation and the mechanism accounting for the processing and compartmentalization of TC10. In this manner, we hope to further developed a molecular understanding of this novel insulin receptor dependent signaling pathway that functions in concert with the PI 3-kinase signaling pathway to induce glucose uptake and GLUT4 translocation in adipocytes.
