DOC2B: a novel syntaxin-4 binding protein mediating insulin-regulated GLUT4 vesicle fusion in adipocytes.

Abstract

Insulin stimulates glucose uptake in skeletal muscle and adipose tissues primarily by stimulating the translocation of vesicles containing a facilitative glucose transporter, GLUT4, from intracellular compartments to the plasma membrane. The formation of stable soluble N-ethyl-maleimide-sensitive fusion protein [NSF] attachment protein receptor (SNARE) complexes between vesicle-associated membrane protein-2 (VAMP-2) and syntaxin-4 initiates GLUT4 vesicle docking and fusion processes. Additional factors such as munc18c and tomosyn were reported to be negative regulators of the SNARE complex assembly involved in GLUT4 vesicle fusion. However, despite numerous investigations, the positive regulators have not been adequately clarified. We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin-4 was assessed by the yeast two-hybrid screening system, immunoprecipitation, and in vitro glutathione S-transferase (GST) pull-down experiments. Cell surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b. Herein, we show that DOC2b, a SNARE-related protein containing double C2 domains but lacking a transmembrane region, is translocated to the plasma membrane upon insulin stimulation and directly associates with syntaxin-4 in an intracellular Ca(2+)-dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca(2+)-interacting domain mutant DCO2b or knockdown of DOC2b inhibited, insulin-stimulated glucose uptake. These findings indicate that DOC2b is a positive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes.