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PubMed2000

Structural analysis of the neuronal SNARE protein syntaxin-1A.

Intracellular trafficking depends on the docking and fusion of transport vesicles with cellular membranes. Central to docking and fusion is the pairing of SNARE proteins (soluble NSF attachment protein receptors) associated with the vesicle and target membranes (v- and t-SNAREs, respectively). Here, the X-ray structure of an N-terminal conserved domain of the neuronal t-SNARE syntaxin-1A was determined to a resolution of 1.9 A using multiwavelength anomalous diffraction. This X-ray structure, which is in general agreement with an NMR structure of a similar fragment, provides new insight into the interaction surface between the N-terminal domain and the remainder of the protein. In vitro characterization of the intact cytoplasmic domain of syntaxin revealed that it forms dimers, and probably tetramers, at low micromolar concentrations, with concomitant structural changes that can be detected by limited proteolysis. These observations suggest that the promiscuity characteristic of pairing between v-SNAREs and t-SNAREs extends to the formation of homo-oligomeric t-SNARE complexes as well. They also suggest a potential role for the neuronal Sec1 protein (nSec1) in preventing the formation of syntaxin multimers.

Antigens, Surface↗
PubMed2002

Membrane topologies of neuronal SNARE folding intermediates.

Assembly of the SNARE complex is essential for neurotransmitter release at synapses. Target plasma membrane SNAREs (t-SNAREs) syntaxin 1A and SNAP-25 form the t-SNARE complex that serves as an intermediate toward final SNARE assembly with vesicle-associated SNARE (v-SNARE). Membrane topologies of syntaxin 1A and the t-SNARE complex were investigated using site-directed spin labeling EPR. EPR analysis revealed that the basic region at the membrane-water interface is unstructured but inserted into the membrane. Such membrane insertion leaves no gap between the t-SNARE core and the membrane. Yet the lack of structure could provide the flexibility necessary for the t-SNARE core. Further, the insertion of the basic interfacial region into the membrane may have profound implications for the mechanism of SNARE-induced membrane fusion.

Amino Acid Sequence↗
PubMed1997

An alpha-helical minimal binding domain within the H3 domain of syntaxin is required for SNAP-25 binding.

The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 binding domain on syntaxin, we have prepared peptides that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25, and then determined the affinities of these peptides for binding to SNAP-25. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism revealed the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis.

Amino Acid Sequence↗
PubMed1998

Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation.

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an Mr of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins: SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2-syntaxin-4, VAMP-2-SNAP23, and SNAP23-syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (<0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.

Animals↗
PubMed1998

Immunohistochemical distribution of epimorphin in human and mouse tissues.

A novel protein epimorphin has been identified as a mesenchymal signal factor. We reported previously ubiquitous expression of epimorphin in normal skin and a significant increased expression in diseased human skin. The present immunofluorescence study was conducted to determine systematically the distribution of epimorphin in adult human organs with an anti-epimorphin monoclonal antibody. Epimorphin was found to be widely distributed in all human organs examined. It was present in the connective tissue adjacent to or around various epithelial tissues, muscles and vessels. In particular, strong staining was present on the endomysium of muscles, the adventitia of blood vessels, along the sinusoidal lining of hepatocytes and connective tissue around epithelial cells, exocrine and endocrine glands. The results suggest that epimorphin may play a key role in maintaining normal tissue structure and interaction between mesenchymal tissue and epithelial tissue in vivo.

Animals↗
PubMedDate not supplied

The hypophysis controls expression of SNAP-25 and other SNAREs in the adrenal gland.

SNAP-25 (Synaptosomal Associated Protein of 25 kDa), in association with two other SNARE (soluble NSF attachment protein receptor) proteins, syntaxin and Vesicle Associated Membrane Protein, VAMP, is implicated in regulated and constitutive exocytosis in neurones and neuroendocrine cells. Our previous studies have shown that it is expressed more by noradrenergic than adrenergic chromaffin cells in the rat adrenal gland. Since certain hormones under hypophyseal control play an essential role in determining chromaffin cell phenotype, the present study examined the effect of hypophysectomy on SNAP-25 expression. Hypophysectomy was found by immunoblotting and RT-PCR analysis to increase adrenal gland SNAP-25, syntaxin-1 and VAMP-2 levels, without modifying the relative expression of SNAP-25 isoforms: immunocytochemistry showed a dramatic increase in SNAP-25 expression in former adrenergic chromaffin cells. Since adrenal glucocorticoids are considerably reduced by hypophysectomy, the effect of corticosterone replacement therapy was investigated. This did not change levels of SNAP-25, syntaxin-1 or VAMP-2. SNARE expression was also unmodified in pheochromocytoma cells treated with a synthetic glucocorticoid. In contrast, subcutaneous injection of hypophysectomized rats with thyroid hormone decreased adrenal SNAP-25, demonstrating the potential importance of the pituitary-thyroid axis. The current data thus demonstrate that the hypophysis exerts an inhibitory control on adrenal gland SNARE proteins. They suggest that glucocorticoids are unlikely to be directly responsible for this but provide evidence that thyroid hormones are implicated in this phenomenon. The putative role of hormonal regulation on SNARE function is discussed.

Adrenal Medulla↗
PubMed2003

Differential expression of syntaxin 1A and 1B by noradrenergic and adrenergic chromaffin cells.

The expression and localization of syntaxin isoforms 1A and 1B in adrenergic and noradrenergic chromaffin cells were examined by both immunoblot analysis and confocal immunofluorescence microscopy. Syntaxin 1A was found in higher levels in noradrenergic cells, whereas syntaxin 1B was similarly expressed in most noradrenergic and adrenergic cells. However, some heterogeneity was observed within each catecholaminergic phenotype. Although the majority of adrenergic cells appeared to express low levels of syntaxin 1A, about 7% was strongly stained for syntaxin 1A. A subpopulation of noradrenergic cells, about 17%, expressed greater levels of syntaxin 1B. Syntaxin 1B labeling showed a punctate appearance in the cytoplasm, whereas syntaxin 1A appeared predominantly localized to the plasma membrane. These data show differences in the exocytotic machinery of the two subtypes of chromaffin cells that may underlie some of the distinct characteristics of adrenaline and noradrenaline secretion.

Adrenal Glands↗
PubMed2004

Syntaxin 1A and receptor for activated C kinase interact with the N-terminal region of human dopamine transporter.

The dopamine transporter (DAT) regulates the extent and duration of dopamine receptor activation through sodium-dependant reuptake of dopamine into presynaptic neurons, resulting in termination of dopaminergic neurotransmission. Using the yeast two-hybrid system, we have identified novel interactions between DAT, the SNARE protein syntaxin 1A, and the receptor for activated C kinases (RACK1). This association involves the intracellular N-terminal domain of human DAT (hDAT). Our data suggest that hDAT may exist as dimers or oligomers and that its protein-protein interactions with syntaxin 1A and RACK1 form functional regulatory complexes that may mediate DAT trafficking through modulation of hDAT phosphorylation by PKC.

Antigens, Surface↗
PubMed1998

The synaptic SNARE complex is a parallel four-stranded helical bundle.

The heterotrimeric synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of the synaptic vesicle-associated membrane protein 2 (VAMP2) and presynaptic plasma membrane proteins SNAP-25 (synaptosome-associated protein of 25,000 Mr) and syntaxin 1A, is a critical component of the exocytotic machinery. We have used spin labeling electron paramagnetic resonance spectroscopy to investigate the structural organization of this complex, particularly the two predicted helical domains contributed by SNAP-25. Our results indicate that the N- and C-terminal domains of SNAP-25 are parallel to each other and to the C-terminal domain of syntaxin 1A. Based on these findings, we propose a parallel four-stranded coiled coil model for the structure of the synaptic SNARE complex.

Animals↗
PubMed1998

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Antigens, Surface↗
PubMed2000

Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex.

Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion.

Amino Acid Sequence↗
PubMed1993

Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.

Three synaptic proteins, syntaxin, SNAP-25 and synaptobrevin, were recently identified as targets of clostridial neurotoxins that irreversibly inhibit synaptic vesicle fusion. Experiments searching for membrane receptors for N-ethylmaleimide-sensitive fusion protein (NSF), which has an important role in membrane fusion, revealed an ATP-dependent interaction of the same three synaptic proteins with NSF and its soluble attachment proteins. Thus, two independent approaches identify syntaxin, synaptobrevin and SNAP-25 as components of the synaptic vesicle fusion machinery, but their mode of action is unclear. We have now discovered a brain protein of relative molecular mass 67,000 (67K) which binds stably to syntaxin. Amino-acid sequencing and complementary DNA cloning revealed that the 67K protein is encoded by the mammalian homologue of the Caenorhabditis elegans gene unc-18. In C. elegans, unc-18 belongs to a group of genes defined by mutations with a paralytic phenotype and accumulations of acetylcholine, suggesting a defect in neurotransmitter release. The binding of the mammalian homologue of unc-18 (Munc-18) to syntaxin requires the N terminus of syntaxin whereas that of SNAP-25 involves a more C-terminal sequence. Our data suggest that Munc-18 is a previously unidentified essential component of the synaptic vesicle fusion protein complex.

Amino Acid Sequence↗
PubMed1997

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

Animals↗
PubMed1995

Functional impact of syntaxin on gating of N-type and Q-type calcium channels.

Rapid and reliable synaptic transmission depends upon the close proximity of voltage-gated calcium channels and neurotransmitter-containing vesicles in the presynaptic terminal. Although it is clear that a local Ca2+ rise conveys the crucial signal from Ca2+ channels to the exocytotic mechanism, little is known about whether communication ever proceeds in the opposite direction, from the release machinery to Ca2+ channels. To look for such signalling, we examined the interaction of various types of voltage-gated Ca2+ channels with syntaxin, a presynaptic membrane protein of relative molecular mass 35,000 which may play a key part in synaptic vesicle docking and fusion and which interacts strongly with N-type Ca2+ channels. Here we report that co-expression of syntaxin 1A with N-type channels in Xenopus oocytes sharply decreases the availability of these channels. This is due to the stabilization of channel inactivation rather than to a simple block or lack of channel expression, because it is overcome by strong hyperpolarization. Deletion of syntaxin's carboxy-terminal transmembrane domain abolishes its functional effect on Ca2+ channels. Syntaxin produced a similar effect on Q-type Ca2+ channels encoded by alpha 1A but not on L-type Ca2+ channels. Thus, the syntaxin effect is specific for Ca2+ channel types that participate in fast transmitter release in the mammalian central nervous system. We hypothesize that, in addition to acting as a vesicle-docking site, syntaxin may influence presynaptic Ca2+ channels, opposing Ca2+ entry where it is not advantageous, but allowing it at release sites where synaptic vesicles have become docked and/or ready for fusion.

Animals↗
PubMed1999

P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

Animals↗