GTPase Regulatory Proteins in Insulin-Stimulated Glucose Transport.

Abstract

Insulin stimulates glucose uptake in muscle and fat by promoting translocation of the facilitative transporter GLUT4 from intracellular compartments to the plasma membrane. Members of the Ras superfamily of small G proteins regulate distinct steps in the GLUT4 trafficking itinerary. The G protein RalA promotes GLUT4 vesicle trafficking to the plasma membrane through its interactions with the actin motor Myo1c and a tethering complex called the exocyst. RalA is activated in response to insulin in 3T3-L 1 adipocytes, however little is known about the proteins regulating this small G protein. An siRNA-based screen for RalA regulators led to the identification of the RalGAP Complex in 3T3-L1 adipocytes. The RalGAP Complex is composed of the regulatory subunit RGC1 (previously identified as KIAA1l219) and the catalytic subunit RGC2 (previously identified as AS250). RGC2 contains a GAP domain at its C-terminus that promotes GTP hydrolysis on RalA and RalB. In 3T3-L1 adipocytes, RGC2 knockdown results in increased basal and insulin-stimulated activation of RalA, demonstrating that this protein negatively regulates RalA. RGC1 binds to RGC2 and is essential for stability of RGC2 protein and regulation for RalA by the Ral GAP Complex. Knockdown of RGC1 or RGC2 increases insulin-stimulated glucose uptake and surface GLUT4 levels, establishing a regulatory role of this complex in glucose transport. RGC2 is directly phosphorylated by Akt2 on at least three residues in response to insulin. Overexpression of active Akt blocks the effects of RGC1/2 on RalA activity, demonstrating that phosphorylation of RGC2 inhibits the complex. Akt2 likely does not affect the catalytic activity of RGC2, but instead results in 14-3-3 binding to and a conformational change in the complex that may occlude it from binding to RalA. The RalGAP GARNL1 serves as an alternative catalytic subunit in the RalGAP complex. This protein is expressed at low levels in adipocytes and does not affect RalA activity or glucose uptake in these cells. However, GARNL1 is expressed in muscle and may regulate RalA activity in response to cellular stimuli in these cells. Together, these data demonstrate that the RalGAP Complex functions as a negative regulator of RalA activity in diverse cellular contexts.