DOC2B: A Novel Syntaxin4 Binding Protein Mediating Insulin-Regulated GLUT4-Vesicle Fusion in Adipocytes

Abstract

Objective: 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 SNARE [soluble N-ethyl-maleimide-sensitive fusion protein (NSF) attachment protein receptor] complexes between VAMP-2 and syntaxin4 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.

Research design and Methods: We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin4 was assessed by yeast two-hybrid screening system, immunoprecipitation and in vitro GST pull-down experiments. Cell-surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b.

Results: 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 syntaxin4 in an intracellular Ca²⁺ dependent manner. Furthermore, this process is essential for triggering GLUT4-vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca²⁺ interacting domain mutant DCO2b or knockdown of DOC2b inhibited insulin stimulated glucose uptake.

Conclusions: These findings indicate that DOC2b is a positive SNARE-regulator for GLUT4 vesicle fusion and mediates insulin stimulated glucose transport in adipocytes.