Tomosyn Is Expressed in β-Cells and Negatively Regulates Insulin Exocytosis

Abstract

E xocytosis is delicately regulated via dynamic protein-protein interactions between different protein components localized to the plasma membrane, the secretory vesicle membrane, and the cytoplasm. According to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis (1,2), the vesicular-SNARE vesicleassociated membrane protein (also called synaptobrevin) interacts with the cognate target-SNAREs syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a core complex (also called SNARE complex) (1). The assembly of SNARE proteins between two opposing membranes and the formation of a core complex have been shown to be the key events that initiate membrane fusion and predict the specificity of vesicle fusion (1,2). That the compartmental specificity of cellular membrane fusion is encoded in SNARE proteins is further provided by the observation that these proteins have distinct localization in a cell (3). However, almost any combination of several members of vesicular-and target-SNARE proteins can form a SDS-resistant protein complex (4,5), suggesting that the interactions between SNARE proteins cannot provide all information for vesicle targeting. Additional specificity may be provided by other molecules that interact with SNARE proteins. An example of such a protein is the well-conserved syntaxin-binding protein Sec1/mammalian homolog of the Caenorhabditis elegans unc-18 gene (Munc-18). There are several Munc-18 isoforms in mammals, which are believed to support different vesicular trafficking events (rev. in 6). Munc-18-1 holds syntaxin in a closed conformation, thereby preventing the binding of SNAP-25 and vesicle-associated membrane protein to syntaxin (7). Moreover, each Munc-18 protein interacts more or less exclusively with one or two syntaxin isoforms, thereby providing further vesicle-targeting specificity (8 -13).The existence of another syntaxin-binding protein, designated tomosyn (tomo ϭ friend in Japanese, syn ϭ syntaxin), has been reported (14). Besides the original tomosyn protein, which has been named m-tomosyn, two further splice variants of tomosyn, designated big (b) and small (s) tomosyn, have been identified (15). The m-and s-tomosyn variants are mainly expressed in the brain, whereas b-tomosyn is found ubiquitously (15). More recently, two distinct genes that drive the expression of seven tomosyn isoforms in the mammalian brain have been described (16). Tomosyn is capable of dissociating Munc-18 from syntaxin 1 and thereby forming a novel complex with syntaxin 1, and synaptotagmin (14). The COOH-terminal domain of tomosyn spans a SNARE motif that allows tomosyn to form a stable complex with syntaxin 1A and SNAP-25 (17,18). Endogenous expression or overexpression of tomosyn has been shown to cause a reduction of Ca 2ϩ -dependent exocytosis (14,19 -23). The structural basis for the inhibitory role of tomosyn in exocytosis has recently been presented (24).In this study, we have investigated isoform expression and cellular localization of tomo...

Mechanisms of biphasic insulin-granule exocytosis – roles of the cytoskeleton, small GTPases and SNARE proteins

“…Moreover, the study by Tomas and colleagues localized the defect to aberrant insulin-granule docking at the plasma membranespecifically, loss of coupling between granules and plasmamembrane syntaxin 1A, which, the authors show, occurs through a novel interaction with the granule protein granuphilin (Tomas et al, 2008). Syntaxin 1A can also associate with the exocytosisinhibiting protein tomosyn and the Munc accessory factor double C2-like domain-containing protein β (Doc2β), although the function of these interactions in biphasic insulin release is untested (Cheviet Zhang et al, 2006). Because Munc18a associates with the syntaxin-1A isoform, which is required for first-phase secretion only, it is hypothesized that Munc18a will also be required specifically for this phase, although this remains to be tested in islets.…”

Platelet secretion and hemostasis require syntaxin-binding protein STXBP5

“…SDF-1, PF4, and angiopoietin 1 were excluded from analysis due to saturation of their respective spots on the array membranes. jci.org Volume 124 Number 10 October 2014 specific for the b-STXBP5 (5′-CTCCGACTTCCGGTTCTTCCTC-3′ and 5′-TTCAGCGTGATGACAAAGGC-3′), m-STXBP5 (5′-CTCCG-ACTTCCGCAAAGATGTC-3′ and 5′-TTCAGCGTGATGACAAAG-GC-3′), and s-STXBP5 (5′-CTCCGACTTCCGATGTGAAAG-3′ and 5′-TTCAGCGTGATGACAAAGGC-3′) isoforms (29,40).…”

“…A specific immunoreactive band was only seen in samples bound to the t-SNARE heterodimers, not in those bound to Munc18a-or Munc18c-containing complexes ( Figure 1B). To determine which STXBP5 isoforms were present in human platelets, RT-PCR analysis was performed using random hexamers and oligo d(T) to prime the reverse transcription step, then using isoform-specific primers for PCR (29,40). Only primers for m-STXBP5 produced a product ( Figure 1C), which suggests that m-STXBP5 is the most abundant isoform in platelets.…”

Syntaxin-binding protein 5 exocytosis regulation: differential role in endothelial cells and platelets

“…In neurons and pancreatic β cells, STXBP5 inhibits neurotransmitter release and insulin secretion, respectively (14,15). Zhu and colleagues demonstrated that STXBP5 also inhibits exocytosis in endothelial cells (8).…”