The regulation of glucose transport by insulin represents the rate-limiting step in glucose utilization and storage, and is known to represent a primary lesion in patients with insulin resistance. The exocyst complex was found in our laboratory to play a role in the regulation of glucose transport, targeting Glut4 vesicles to sitesof docking and fusion in fat cells. The overall goal of this proposal is to explore the molecular details of exocyst assembly and recognition, focusing on the key events that define its role in insulin-stimulated glucose transport both in vivo and in vitro. We propose that insulin stimulates the activity of Akt2, which phosphorylates the Ral GTPase-activating proteins (GAP) complex (RGC), resulting in sequestration from its target G protein, RalA. We propose that RalA is thus activated, and then directs Glut4 vesicles along the final steps of the journey to the exocyst, where it binds to the exocyst components Sec5 and Exo84. Once bound to RalA, both Exo84 and Sec5 undergo phosphorylation, in the process releasing RalA from the exocyst and readying the vesicle for fusion. We propose to explore this hypothesis by focusing on four specific aims: 1) understanding the physiological role of the RGC in glucose homeostasis by investigating conditional knockout mice that we have created; 2) investigating the physiological role of RalA in glucose homeostasis using a combination of knockout mice and selective Ral inhibitors; 3) understanding how a G protein cascade operates in adipocytes through recruitment of the Ral guanine nucleotide exchange factor (GEF) Rgl2 by the upstream G protein Rab10; and 4) investigating the important role of Exo84 phosphorylation by the protein kinase TBK1. We anticipate that these data will present a clearer picture of the role of the exocyst in the regulation of Glut4 trafficking, docking and fusion.
Glucose transport is the rate-limiting step in hormonally-regulated energy storage in muscle and fat cells, and is mediated by the glucose transporter Glut4. We will evaluate the role of the exocyst complex in this process, an evolutionarily conserved, eight protein targeting complex that ensures the localization of Glut4 in the cell.
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