DOC2B: A Novel Syntaxin-4 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 soluble N-ethyl-maleimidesensitive 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.RESEARCH DESIGN AND METHODS-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.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 syntaxin-4 in an intracellular Ca 2ϩ -dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca 2ϩ -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.

GLUT4 exocytosis

“…Double C2-like domains beta (DOC2B), a protein that contains two C2 domains, is localised to small punctate structures that are scattered throughout the cytoplasm in adipocytes; from these it translocates to the plasma membrane and binds to syntaxin-4 in response to insulin in a Ca 2+ -dependent manner. Reducing DOC2B levels by RNA interference impairs insulin-dependent glucose transport in adipocytes (Fukuda et al, 2009). C2 domains insert into membranes and promote membrane curvature, thus further aiding fusion (McMahon et al, 2010).…”

The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets

“…While the requirement of Ca 2C for Rip11 binding to acidic phospholipids has yet to be determined, in adrenal chromaffin cells, Doc2b is recruited to the cell surface in a Ca 2C -dependent manner where it functions as a priming factor and increases the number of fusion-competent vesicles (Groffen et al 2004, Friedrich et al 2008. A recent study in 3T3-L1 adipocytes has demonstrated that Doc2b is recruited to the plasma membrane in an insulinand Ca 2C -dependent manner where it associates with syntaxin4 (Fukuda et al 2009). The recruitment of Doc2b was found to be necessary for GLUT4 vesicle fusion as both a mutant deficient in Ca 2C binding and the siRNA-mediated depletion of Doc2b from adipocytes inhibited insulinstimulated glucose uptake (Fukuda et al 2009).…”

“…A recent study in 3T3-L1 adipocytes has demonstrated that Doc2b is recruited to the plasma membrane in an insulinand Ca 2C -dependent manner where it associates with syntaxin4 (Fukuda et al 2009). The recruitment of Doc2b was found to be necessary for GLUT4 vesicle fusion as both a mutant deficient in Ca 2C binding and the siRNA-mediated depletion of Doc2b from adipocytes inhibited insulinstimulated glucose uptake (Fukuda et al 2009).…”

“…This process has recently been shown to involve the insulin-stimulated phosphorylation of Munc18c on Tyr219, which is thought to induce a change in the binding specificity of Munc18c from syntaxin4 to Doc2b (Jewell et al 2008), a double C2 domain-containing protein that has been identified as a positive regulator of exocytic vesicle fusion in both adipocytes and adrenal chromaffin cells (Ke et al 2007, Friedrich et al 2008, Sano et al 2008, Fukuda et al 2009). Other syntaxin4 binding proteins that appear to function as negative regulators of GLUT4 vesicle fusion include Synip (Min et al 1999) and Tomosyn ( (Widberg et al 2003); these are not included in Fig.…”

DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain β (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer

“…Binding of calcium to DOC2B significantly increases its affinity toward phospholipids, leading to translocation of proteins from the cytosol to plasma membrane (8). Recently, DOC2B was shown as a posi-tive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes as well as a regulator for delayed insulin secretion in MIN6 cells (9). It is involved in the deformation of synaptic membranes during synaptic vesicle exocytosis (10,11).…”