Fluorescence Resonance Energy Transfer Reports Properties of Syntaxin1A Interaction with Munc18-1 in Vivo

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Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins (1). Although the primary molecular entities in the release pathway have been identified, the exact mechanics of synaptic vesicle fusion and its precise regulation are still not established. Central to the fusion process is the transient formation of SNARE 4 core complexes that include the target membrane SNARE proteins syntaxin 1A and SNAP25 and the vesicle SNARE protein synaptobrevin/VAMP (2-4). A SNARE core complex is a highly stable, four-␣-helix parallel bundle consisting of one SNARE motif from each of syntaxin 1A and synaptobrevin/VAMP, and two SNARE motifs from SNAP25 (5, 6). Although these proteins alone are sufficient to induce a slow fusion when reconstituted into liposomes (7), additional proteins are necessary to establish the properties that describe fast, Ca 2ϩ -dependent neurotransmitter release (8). For example, assembly of SNARE core complexes is subject to temporal and spatial regulation by a variety of protein families, including Rab-GTPases (9 -13), Sec/Munc18s (14 -16), exocyst tethering complexes (17-20), and Munc13s (21-24). In addition, recent evidence suggests that the temporal and spatial availability...

Section: Expression and Targeting Of Fluorescently Tagged Tomosyn Symentioning

confidence: 74%

Section: Confocal and Conventional Fluorescence Microscopy Of Ecfp-anmentioning

confidence: 99%

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Gladycheva

Lam

Liu

et al. 2007

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“…Confocal Microscopy and Measurement of FRET-Fluorescence imaging and FRET analysis were performed using an LSM 510 confocal microscope according to the protocol as previously described by Liu et al (30). Cells were grown on polylysine-coated glass coverslips and fixed with 3% paraformaldehyde.…”

Section: Methodsmentioning

confidence: 99%

Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

Zhang

Tan

Zhang

et al. 2008

Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg 9 -bradykinin or desArg 10 Carboxypeptidase M (CPM) 2 was discovered as a glycosylphosphatidylinositol (GPI)-anchored membrane protein with B-type carboxypeptidase activity (1-3) and is a member of the "regulatory" or carboxypeptidase N/E subfamily of metallocarboxypeptidases (4 -8) based on its cDNA sequence (9), genomic structure (10), and x-ray crystal structure (11). The overall structure of CPM is composed of a 295-residue N-terminal catalytic domain, followed by an 86-residue conical ␤-sandwich (transthyretin-like domain) and a unique 25-residue extension to which the GPI anchor is post-translationally attached (11).CPM cleaves only C-terminal Arg or Lys residues, and some of its endogenous substrates include bradykinin, anaphylatoxins C3a, C4a, and C5a, Arg-or Lys-enkephalins, epidermal growth factor, and hemoglobin (2, 7, 12, 13). CPM preferentially cleaves C-terminal Arg as exemplified by the kinetics with Arg 6 -Met 5 -enkephalin (K m ϭ 46 M, k cat ϭ 934 min Ϫ1 ) versus Lys 6 -Met 5 -enkephalin (K m ϭ 375 M, k cat ϭ 663 min Ϫ1 ) (2). Bradykinin exhibits the lowest K m (16 M) of any CPM substrate tested (2), a concentration that is still much higher than the typical physiological concentration of this peptide in the nanomolar range. However, peptidases in vivo typically work at substrate concentrations far below the K m as exemplified by angiotensin I-converting enzyme (ACE), which has the same relatively high K m (16 M) with angiotensin I (14), a major physiological substrate. The development of ACE inhibitors as effective agents for treating hypertension and cardiovascular diseases was based in large part on the critical role of this enzyme in converting angiotensin I to II in vivo (15).The kinin peptides bradykinin and kallidin (Lys-bradykinin) are generated by the proteolytic action of plasma or tissue kallikrein on high or low molecular weight kininogen (16,17). Removal of the C-terminal Arg from bradykinin or kallidin inactivates these peptides as agonists of the constitutively expressed B2 receptor (B2R) (16,17). However, this conversion is a required processing step to generate desArg 9 -bradykinin or des-Arg 10 -kallidin (16, 18), metabolites that have a variety of biological activities mediated by specific activation of a different B1 receptor (B1R) whose expression is induced by inflammatory mediators (19,20). Thus, CPM acts as a cell surface processing enzyme to generate des-Arg-kinin agonists for the B1R, and without this catalytic conversion, B1R signaling could not occur. This is of potential importance in inflammatory or pathological responses. For example, B1R activation stimulates extracellular signal-regulated kinase phosphorylation, prostaglandin production (20), and inducible nitric-oxide synthase-mediate...

“…To generate N-terminal fluoroprotein-labeled constructs, the following cDNAs were subcloned into the SalI-XbaI sites of pDNR-dual for use with the Cre recombinase-mediated Creator System (Clontech): rat Munc18a (pGex-KG-Munc18a), rat syntaxin 1A (pGEX-syntaxin 1A 11 ), rat Munc18c (pcDNA3-FLAG-Munc18c), human syntaxin 4 (pcDNA4/TO/syntaxin 4-Myc 2 -His), and human SNAP23 (pcDNA3-SNAP23) (gifts from J. Pevsner, R. Scheller, J. Pessin, and T. Weimbs (syntaxin 4 and SNAP23), respectively). The recipient vectors pLoxP-ECFP-C1 and pLoxP-EcYFP-C1 (Q39M mutant of pEYFP-C1; citrine) were generated and mutated to their monomeric forms (A206K) from pLoxP-EGFP-C1 (Clontech) as described previously (17). To prepare pLoxP-mRFP1-C1, the cYFP sequence from pLoxP-EcYFP-C1 was replaced with mRFP1 (monomeric form) from pRSETB-mRFP1 (a gift from R. Tsien) by PCR cloning.…”

Section: Methodsmentioning

confidence: 99%

“…The open conformation facilitates SNARE-SNARE pairing resulting from accessibility of the SNARE motif. The closed (SNARE pairing inactive) conformation of syntaxin 1A is stabilized by interaction with Munc18a (15), precluding interactions with other SNAREs (16,17). Thus, the association of Munc18 suggests a mechanism for the regulation of the syntaxin SNARE motif through the control of syntaxin conformation.…”

mentioning

confidence: 95%

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Merrins

Chang

Lam

et al. 2007

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In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4 LE -SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4 LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4 LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.Soluble N-ethylmaleimide-sensitive factor (NSF) 3 attachment protein receptors (SNAREs) are central to vesicle fusion events in all eukaryotes. SNARE proteins anchored to both transport vesicles and their target membranes overcome the forces necessary for fusion by binding with high affinity in a ternary core complex (1). Although formation of SNARE core complexes is sufficient for membrane fusion (2, 3), essential regulatory proteins of the Sec1/Munc18 (SM) family are believed to control SNARE complex formation. Seven vertebrate SM gene family members (Munc18a/b/c, Sly1, Vps45, and Vps33a/b) have been described, which affect membrane trafficking in distinct subcellular pathways, predominantly through their association with specific syntaxin Q-SNAREs (4).Of the varied SM-syntaxin partners, Munc18c-syntaxin 4 interactions are believed to exert a critically important role in the temporal control of insulin-stimulated GLUT4 storage vesicle exocytosis in muscle and adipose tissue (5-8). The delivery of GLUT4 to the cell surface occurs by distinct signaling processes: 1) GLUT4 vesicle recruitment to the plasma membrane, followed by 2) phosphatidylinositol 3-kinase-dependent vesicle docking and fusion mediated by syntaxin 4-containing SNARE complexes (9, 10). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 ad...