Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting ␤-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca 2؉ -induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary ␤-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca 2؉ -dependent insulin secretion from primary ␤-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.Exocytosis of insulin from pancreatic ␤-cells has been suggested to be mediated by the same core fusion machinery that controls all membrane fusion events in organisms ranging from yeast to human (1-3). The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) 1 proteins are essential components of this machinery. Proteins localized in the membrane of the transported vesicle (v-SNAREs) specifically interact with proteins in the target membrane (t-SNAREs). In neurotransmitter release from synaptic vesicles, the plasma membrane-associated proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) interact with the vesicular component synaptobrevin/vesicular-associated membrane protein (VAMP) (reviewed in Refs. 4 -6). Several synaptic proteins regulating neuronal exocytosis including syntaxin, SNAP-25, VAMP, and Munc18-1 have also been identified in pancreatic ␤-cells, supporting the idea that the molecular machinery regulating insulin secretion is similar to that of neurotransmitter release from synaptic vesicles (7-11).There are a series of discrete biochemical steps leading to trans-SNARE complex formation and vesicular fusion. The vesicles need to be transported and targeted to the cell surface where they are docked, primed, and finally fused with the plasma membrane. Munc18-1, a member of the sec1/Munc18 protein family has emerged as a critical regulator of exocytosis (12)(13)(14). Indeed, Munc18-1 is shown to be important for vesicle trafficking and essential for synaptic transmission since both synaptic transmission and spontaneous neurotransmitter release is abolished in neocortical neurons from Munc18-1-null mouse mutants (15). Albeit essential for regulated exocytosis, it has been demonstrated both in pancreatic ␤-cells and in neuronal systems, that Munc18-1 may also serve as a negative regulator of secretion (11,16,17). Munc18-1 binds to syntaxin 1 and might thus negatively regulate syntaxin 1 function if the expressi...

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...

The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

OBJECTIVENephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function.RESEARCH DESIGN AND METHODSNephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin–transfected cells was used to study nephrin endocytosis.RESULTSNephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin–transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release.CONCLUSIONSOur data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.

Cytosolic free Ca 2؉ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic ␤ cell. A number of Ca 2؉ -binding proteins have been implicated in this process. Here, we define the role of the Ca 2؉ -binding protein neuronal Ca 2؉ sensor-1 (NCS-1) in insulin secretion. In pancreatic ␤ cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (PI) 4-kinase ␤ activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca 2؉ -dependent activator protein for secretion present in ␤ cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic ␤ cell. We also demonstrate that NCS-1 and its downstream target, PI 4-kinase ␤, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.insulin ͉ phosphoinositides ͉ islet ͉ secretion ͉ Ca 2ϩ -dependent activator protein for secretion N euronal Ca 2ϩ sensor-1 (NCS-1) belongs to the family of EF-hand Ca 2ϩ -binding proteins and is mainly expressed in neuronal and neuroendocrine cells, where it enhances neurotransmission and Ca 2ϩ -dependent exocytosis (1-5). NCS-1 interacts with and regulates the activity of phosphatidylinositol 4-kinase ␤ (PI4K␤) (6-9). The members of the PI4K family catalyze the first step in the synthesis of PI 4,5-bisphosphate [PI(4,5)P 2 ], which has recently emerged as an important regulator of Ca 2ϩ -dependent secretion (10-12). Both a PI transfer protein (13) and a PI4P 5-kinase (14) are required for regulated exocytosis of dense core granules. In addition, the presence of synaptic vesicle and dense core granule-associated PI4K activity is essential for exocytosis (15,16). These data imply that generation of PI(4,5)P 2 is an important step in the event of secretion.In pancreatic ␤ cells, glucose dose-dependently increases ATP levels and decreases ADP levels (16). The resulting rise in the ATP at the expense of ADP is an important regulator of the two major signaling pathways involved in glucose-induced insulin secretion. The first of these pathways uses ATP-sensitive K ϩ channels to couple glucose metabolism with electrical activity, Ca 2ϩ influx, and initiation of insulin secretion. The second pathway is exerted at the level of granule priming and regulates the ␤ cell secretory capacity by modulation of the granules' release competence (17). The ␤ cell contains Ϸ10,000 insulincontaining secretory granules (18). Interestingly, as many as Ϸ5% of the granules are docked below the membrane. The readily releasable pool (RRP), defined by functional measurements, represents a subset (50-100 granules) of the docked pool (19). Granules belongin...

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in ␤-cell stimulus-secretion coupling. We show that pancreatic ␤-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. To maintain glucose homeostasis in the body, the pancreatic ␤-cell must in a controlled way produce, store, and secrete insulin in response to appropriate stimuli (1). Regulated membrane fusion resulting in insulin exocytosis in ␤-cells is managed by the same core SNARE 3 (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) fusion machinery that mediates release of chemical signals at highly specialized central neuronal synapses (2-4). Three conserved SNARE proteins, VAMP2, syntaxin 1A, and SNAP-25, connect vesicles with the plasma membrane by forming an exceptionally stable protein complex that holds the intrinsic, but slow, capability to perform lipid bilayer fusion (5). Except for the SNARE proteins, additional cooperating proteins are critical to guarantee speed and accuracy of diverse membrane fusion events occurring in excitable cells. Such a key accessory regulator in synaptic transmission is Munc18-1, a member of the Munc18 (mammalian homologue of the unc-18 gene) family (6). In addition to Munc18-1 (with two alternatively spliced variants, Munc18-1a and Munc18-1b), Munc18-2 and Munc18-3 isoforms have also been identified (7-9). Munc18-2, also named Munc18b or muSec1 (mammalian ubiquitously Sec1), is the closest homologue of Munc18-1, showing 63% amino acid sequence identity (7).The 67-kDa hydrophilic Munc18-1 protein has been shown to be necessary not only for vesicle exocytosis but also for docking of vesicles to the plasma membrane (10). Indeed, in the absence of Munc18-1 in neuronal cells, neither evoked nor spontaneous neurotransmission can operate (6). However, besides the reported positive effect of Munc18-1 in regulated exocytosis (6, 11-13), Munc18-1 has also been identified as a negative regulator of insulin secretion (14, 15). Still, Munc18-1 is primarily considered to be a neuronal protein, whereas the homologue Munc18-2 has a wider tissue expression profile. For instance, Munc18-2 is prominent in epithelial cells (16) and has been identified as a binding partner of Cab45, a Ca 2ϩ -binding protein prominent in pancreatic acini (17). Recently Cab45/ Munc18-2 was a...

Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant tumor syndrome, with the presence of tumors in parathyroid, pancreatic, and anterior pituitary. The tumor suppressor gene MEN1, located on chromosome 11q13, encodes a 610 amino acid, 68-kDa protein, menin. Menin is conserved among species but has no similarity with any known protein. To investigate how the expression is regulated in both man and mouse, we assayed a greater than 1-kb region upstream of the second exon for promoter activity in luciferase reporter vectors. The basic promoter was located closely upstream the most commonly expressed first exon. The region further upstream modified the activity. Repetitive elements of the short interspersed/Alu type covered the entire human upstream regulatory region and were the only apparent motif in common with its murine ortholog. Previous studies have indicated a compensatory induction of the second allele because of inactivation of the first allele. We found that overexpression of menin in an inducible cell culture system down-regulated the proximal promoter. In response to down-regulation of MEN1 expression by RNA interference, the regulatory region activated the promoter in a compensatory manner. Our data confirm that the expression of the MEN1 gene is regulated by a feedback from its product menin.