Signaling pathways involved in insulin-stimulated glucose transport

Insulin is a key hormone secreted from pancreatic beta cells that is essential for glucose homeostasis. The hormone rapidly stimulates glucose uptake into muscle and adipocytes, via the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. Although the cellular dynamics of GLUT4 trafficking are well studied, our understanding of the signaling pathways linking the insulin receptor to GLUT4 translocation remains incomplete. While tyrosine phosphorylation of the IRS family of insulin receptor substrates and the subsequent activation of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) is required, numerous studies indicate that it is not sufficient for this action of insulin.; This thesis describes a new PI 3-kinase independent pathway that is specifically activated in response to insulin, downstream of the CAP/Cbl axis, which is also essential for the stimulation of GLUT4 translocation by insulin. Upon tyrosine phosphorylation in response to insulin, Cbl translocates with CAP to a lipid raft subdomain in the plasma membrane, due to the interaction of CAP with flotillin. Phospho-Cbl then binds to CrkII, in the process recruiting the Crk-associated protein C3G into lipid rafts. The Rho GTPase TC10 in lipid rafts is in turn activated by C3G due to its close proximity.; This thesis also characterizes the mouse homologue of TC10 (TC10α) and another TC10-like Rho GTPase (TC10β) cloned from 3T3-L1 adipocytes. Surprisingly, both TC10α and TC10β reside in 3T3-L1 adipocytes, and expression of tagged forms of the proteins revealed their localization in caveolin-enriched lipid rafts. In addition, both GTPases can be activated by insulin.; A novel Synip-interacting Rho GAP protein (TCGAP) was isolated from a yeast two-hybrid screen. This TCGAP specifically interacts with the WW domain of Synip, the Syntaxin 4 interacting protein that may regulate v- and t-SNARE interactions that are crucial to GLUT4 vesicle docking and fusion. The constitutive association between TCGAP and Synip facilitates the dissociation of Synip from Syntaxin 4 in response to insulin. In addition to the specific binding to Synip, TCGAP also interacts with Rho GTPases in a GTP-dependent manner through its Rho GAP domain. The substrate specificity of the Rho GAP activity of TCGAP correlates well with its in vitro binding affinity toward Rho GTPases.; Elucidation of the events downstream of TC10 activation and inactivation may help further the understanding of the signaling pathways critical to insulin action.