Coordination of Cell Signaling and Transport Machineries in Insulin-Stimulated Glucose Transport

Abstract

Insulin-stimulated glucose transport is the rate-limiting step in glucose disposal and utilization. Insulin increases glucose uptake in fat and muscle through the translocation of the insulin-responsive glucose transporter Glut4 to the plasma membrane. Although our understanding of the pathways governing this process remain incomplete, small GTPases have been implicated as “molecular switches” that operate at the crossroads of insulin signaling and Glut4 translocation. This thesis elucidates the role of RalA, a small GTPase that regulates an octameric vesicle-tethering complex known as the exocyst during Glut4 trafficking.

Initial studies on the cell cycle revealed that both RalA and the exocyst are involved in trafficking through the recycling endosome. Loss of RalA or the exocyst led to a specific blockade in cell abscission, the very last stage of cytokinesis, implying the involvement of these proteins in a subset of transport events under tight regulation.

Glut4 traffics through the recycling endosome in adipocytes. We found that RalA resides on the Glut4 vesicles and interacts with the exocyst in insulin-responsive cells. Insulin activates RalA in a PI-3 kinase-dependent manner. Disruption of RalA function led to inhibition of insulin-stimulated glucose transport, as did loss of the exocyst. Furthermore, RalA also binds to Myo1c, a molecular motor previously implicated in Glut4 trafficking. This interaction is modulated by Calmodulin, which functions as the light chain for Myo1c during insulin-stimulated glucose transport. The data suggested a dual role for RalA in insulin action, as a cargo receptor for Myo1c and a signal to unify the exocyst.

The architecture of the exocyst complex was further dissected with RalA mutants uncoupled from one branch of its effectors, including the two exocyst subunits Sec5 and Exo84. We found that both subunits are required for exocyst function in glucose transport; however, they belong to different branches of the exocyst complex that also contain overlapping subunits. Furthermore, three exocyst subunits Sec8, Sec6, and Sec5 form a sub-complex that targets RalA-localized vesicles.

Taken together, these data suggest that RalA integrates upstream signaling from the insulin receptor to mobilize downstream transport machineries, leading to the specificity required for the actions of insulin.