Molecular Cloning of an Isoform of Doc2 Having C2-like Domains

The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2␤ as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2␤ was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2␤-Munc18c binding to be direct and complexes to be devoid of Glucose homeostasis is maintained by a balance of insulin secretion and insulin action. Insulin is secreted from islet beta cells filled with mature insulin-containing granules which traffic to and fuse with the cell surface upon stimulation by elevated blood glucose. Upon detection of insulin and elevated circulating glucose levels by the skeletal muscle and adipose tissues the intracellular vesicles containing the insulin-responsive glucose transporter GLUT4 translocate to the plasma membrane and facilitate glucose uptake into the cell (1, 2). These "vesicle exocytosis" events are known to be regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 2 proteins (2, 3). Vesicle exocytosis entails the specific pairing of a vesicle-associated membrane protein v-SNARE (VAMP) with a binary cognate receptor complex t-SNARE composed of SNAP-25/23 and Syntaxin proteins at the target membrane to form the SNARE core complex (3, 4). SNARE protein functions are further regulated by interaction with the Sec1/Munc18 (SM) secretory proteins, of which three plasma membrane-localized homologues exist in mammalian cells: Munc18a, Munc18b, and Munc18c (5, 6). However among these, only the Munc18c isoform can bind with and regulate the t-SNARE protein Syntaxin 4 (7, 8), and Syntaxin 4 is the singular functional Syntaxin isoform in insulin-stimulated GLUT4 vesicle exocytosis (9 -11) and one of two functional Syntaxin isoforms identified in glucose-stimulated insulin exocytosis (12-15). Whereas we and others (11, 16) have shown that either depletion or overexpression of Munc18c in vivo dramatically alters glucose homeostasis via disruption of skeletal muscle GLUT4 translocation and pancreatic islet function, the mechanism by which Munc18c regulates these particular exocytotic events remains unknown.Crystallographic and NMR studies support the concept that the Munc18 protein may keep its cognate Syntaxin in a "closed" conformation (17)(18)(19). Very recent studies further suggest that the Munc18 protein assists the transition of Syntaxin to the open state, possibly by stabilizing a labile transition half-open state of Syntaxin (20,21). Previous studies of Munc18c-Syntaxin 4 kinetics in 3T3L1 adipocytes ar...

Section: Doc2␤ and Munc18c Associate In Min6 Betamentioning

confidence: 99%

Doc2β Is a Novel Munc18c-interacting Partner and Positive Effector of Syntaxin 4-mediated Exocytosis

Thurmond

2007

“…To date, four different types of C-type tandem C2 proteins (i.e. the synaptotagmin (Syt) family (3)(4)(5)9), rabphilin-3 (10), the Doc2 family (11)(12)(13)(14), and the Syt-like protein (Slp) family (8,15)) have been reported, and they are distinguished from each other by their unique N-terminal domains (8,15). The members of the Slp family are coded by at least four distinct genes in mouse and human and have several alternatively splicing isoforms (15): Slp1/JFC1 (8,16), Slp2-a-d, Slp3-a/b (8,15), and Slp4/granuphilin-a (17).…”

mentioning

confidence: 99%

The Slp Homology Domain of Synaptotagmin-like Proteins 1–4 and Slac2 Functions as a Novel Rab27A Binding Domain

Kuroda¹,

Fukuda²,

Ariga³

et al. 2002

205

261

rab27A, which encodes a small GTP-binding protein, was recently identified as a gene in which mutations caused human hemophagocytic syndrome (Griscelli syndrome) and ashen mice, which exhibit defects in melanosome transport as well as in regulated granule exocytosis in cytotoxic T lymphocytes. However, little is known about the molecular mechanism of Rab27A-dependent membrane trafficking or the specific effector molecules of Rab27A. In this study, we discovered that the Slp (synaptotagmin-like protein) homology domain (SHD) of Slp1-3 and Slac2-a/b specifically and directly binds the GTP-bound form of Rab27A both in vitro and in intact cells but not of the other Rabs tested (Rab1, Rab2, Rab3A, Rab4, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). Immunocytochemical analysis revealed that Slp2 (or Slp1) colocalized with Rab27A in the melanosomes of melanoma cells. Slp2 and Rab27A were distributed to the periphery of the cells (especially at the dendritic tips) in the wild-type melanoma cells, whereas they accumulated in the perinuclear region in the melanosome transport-defective cells (S91/Cloudman). These results strongly indicated that the SHD of Slp1-3 and Slac2 functions as an in vivo Rab27A binding domain.

“…DOC2A and DOC2B are both expressed in the brain, and in addition DOC2B is found in several peripheral tissues (6,7). In subcellular fractionations, DOC2A and -B copurify with secretory vesicles, although they lack a transmembrane domain.…”

mentioning

confidence: 99%

Ca2+-induced Recruitment of the Secretory Vesicle Protein DOC2B to the Target Membrane

Groffen

Brian

Dudok

et al. 2004

Ca 2؉ -dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca 2؉ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca 2؉ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca 2؉ -dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca 2؉ -induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca 2؉ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca 2؉ -induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.Ca 2ϩ -induced exocytosis is a widely conserved mechanism of importance for a broad range of secretory systems, such as the release of neurotransmitters and neuropeptides in the central and peripheral nervous system. To attain fusion competence, secretory vesicles must first dock at the target membrane and undergo a process called priming. The release of fusion-ready vesicles is coupled to the opening of Ca 2ϩ channels in the plasma membrane, producing a transient high Ca 2ϩ concentration (10 -100 M for ϳ1 ms) in close proximity to the channels. The inward Ca 2ϩ current then diffuses into the cytoplasm, resulting in a residual concentration in the range of 1 M during tens of seconds. It is widely accepted that this residual Ca 2ϩ concentration induces short term changes in exocytotic strength (e.g. see Refs. 1 and 2), although the mechanisms that contribute to this phenomenon remain largely unclear. As a result, the secretory strength of neuronal and endocrine release sites is modulated in an activity-dependent way. This phenomenon is important to avoid vesicle depletion during repetitive stimulation and is furthermore considered in neurons to contribute to memory and learning.Although Ca 2ϩ is the most important messenger after high frequency activation of the release site, exocytotic potentiation can also be induced by diacylglycerol (synthesized by members of the phospholipase family) or by its phorbolester analogues. Diacylglycerol has multiple intracellular targets (e.g. protein kinase C isoforms), but the potentiating effect in regulated exocytosis is mediated by the priming factor Munc13-1. Munc13-1-deficient mice die shortly after birth and show a defect in synapt...