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PubMed1994

Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking.

Intracellular vesicles destined to fuse with the plasma membrane and secrete their contents must have a mechanism for specifically interacting with the appropriate target membrane. Such a mechanism is now suggested by the demonstration of specific interaction between vesicular proteins and plasma membrane proteins. The vesicle-associated membrane proteins (VAMPs) 1 and 2 specifically bind the acceptor membrane proteins syntaxin 1A and 4 but not syntaxin 2 or 3. The binding site is within amino acids 194 to 267 of syntaxin 1A, and the approximate equilibrium dissociation constants is 4.7 x 10(-6) molar. These data suggest a physical basis for the specificity of intracellular vesicular transport.

Amino Acid Sequence↗

PubMed2003

A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly.

The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca(2+)-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.

Binding Sites↗

PubMed2002

Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691-36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming endocytic material.

Amino Acid Sequence↗

PubMed1994

Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.

omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.

Amino Acid Sequence↗

PubMed2003

Acid-sensing ion channels in malignant gliomas.

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.

Acid Sensing Ion Channels↗

PubMed2001

Loss of heterozygosity and microsatellite instability at chromosomal sites 1Q and 10Q in morphologically distinct regions of late stage prostate lesions.

PURPOSE: We investigated the incidence of loss of heterozygosity (LOH) and microsatellite instability in sporadic prostate cancer and surrounding tissue at loci encompassing the HPC1 and PTEN genes. MATERIALS AND METHODS: Surgical specimens from 63 patients with sporadic stage T3 or T4 prostatic adenocarcinoma were analyzed for LOH and microsatellite instability. Microdissected tissue included morphologically normal foci, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma. LOH analysis was performed using 4 microsatellite markers that map in the region of the 1q24 to 25 locus of the putative prostate cancer susceptibility gene HPC1 and 4 that map in the region of the 10q23 locus of the PTEN gene. RESULTS: The incidence of LOH on 10q was consistent with that previously reported in prostatic tumors. LOH associated with the PTEN locus was recorded in morphologically normal foci, BPH and adenocarcinoma. Sequence analysis of PTEN in a limited number of lesions revealed mutations in nontumor and tumor tissue. Analysis of the DS10215 locus showed significant LOH in tumor but not in benign tissue, suggestive of a tumor suppressor gene in this region associated with prostatic neoplastic progression. In contrast, no significant LOH was observed in the same tissues at 4 loci on chromosome 1q. In this study we recorded elevated levels of microsatellite instability in benign prostatic tissue with an additional increase associated with prostatic adenocarcinoma. CONCLUSIONS: The low incidence of LOH in the region of the HPC1 locus in all prostate lesions studied suggests that this putative hereditary prostate cancer susceptibility locus does not appear to have a role in sporadic prostate cancer, at least not in the context of LOH. In contrast, analysis of the same tissues for LOH at chromosome 10q confirmed frequent alterations in this region linked to late stage prostate cancer. PTEN mutations in microdissected morphologically normal and BPH tissue showed alterations in nontumor tissue surrounding adenocarcinoma. Microsatellite instability was increased in adenocarcinomas over an elevated background recorded in surrounding tissues.

Adenocarcinoma↗

PubMed2001

African-American heredity prostate cancer study: a model for genetic research.

A genome-wide scan of high-risk prostate cancer families in North America has demonstrated linkage of a particular marker to Chromosome 1q (HPC1). An even greater proportion of African-American families have shown linkage to HPC1. Therefore, investigators at the National Human Genome Research Institute (NHGRI) in collaboration with Howard University and a predominantly African-American group of urologists established the African-American Hereditary Prostate Cancer (AAHPC) Study Network to confirm the suggested linkage of HPC in African Americans with a gene on Chromosome 1. Blood samples from recruited families were sent to Howard University for extraction of DNA. The DNA was sent to NHGRI at NIH where the genotyping and genetic sequence analysis was conducted. Genotype data are merged with pedigree information so that statistical analysis can be performed to establish potential linkage. From March 1, 1998, to June 1, 1999, a total of 40 African-American families have been recruited who met the study criteria. Preliminary results suggest that racial/ethnicity grouping may affect the incidence and extent of linkage of prostate cancer to specific loci. The importance of these findings lays in the future treatment of genetic-based diseases.

Aged↗

PubMed2003

False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions.

The techniques of co-immunoprecipitation and immunocytochemical co-labelling are classically used to identify protein-protein interactions. We have used an antibody to the rat small conductance calcium-activated potassium channel subtype 1 (rSK1) to immunoprecipitate proteins from rat brain. A 35 kDa protein was recognized by two monoclonal antibodies to syntaxin 1 and a polyclonal antibody to syntaxin 1A, but not by antibodies to syntaxins 2, 3 or 4. These data suggested that syntaxin 1A is specifically associated with rSK1 in rat brain. A GST construct of the carboxyl terminus of rSK1 was able to pull-down syntaxin 1A from rat brain. Immunocytochemistry showed somatic labelling for both rSK1 and syntaxin 1A in acutely dissociated hippocampal CA1 neurons, confirming that these proteins could interact in vivo. However, control immunoprecipitations showed that antibodies to eight potassium channels could also immunoprecipitate syntaxin, even though some of these channels would not be expected to reside in the same subcellular compartment. Mock immunoprecipitations and pull-down assays showed that syntaxin 1 could directly interact with sepharose and agarose resins. Hence immunoprecipitation and pull-down assays do not provide evidence that syntaxin is specifically associating with a protein, placing doubt on a number of reported interactions with syntaxin 1A.

Animals↗

PubMed2003

Intracellular accumulation of pIgA-R and regulators of transcytotic trafficking in cholestatic rat hepatocytes.

Bile duct ligation (BDL) impairs basolateral-to-apical transcytosis in hepatocytes, causing accumulation of transcytotic carriers for the polymeric IgA receptor (pIgA-R) and redistribution of secretory component (SC) from bile to blood. To gain insight into the mechanisms regulating transcytosis and the pathophysiology of cholestasis, we investigated nascent protein trafficking in control and BDL livers using cell fractionation in the context of in vivo pulse-chase experiments and immunoblot analysis. Control and cholestatic hepatocytes trafficked [35S]-labeled serum proteins and the pIgA-R along the secretory pathway with identical kinetics. However, BDL impaired transcytosis, causing (1) accumulation of the pIgA-R, rab3D, rab11a, and other candidate regulators of apical-directed secretion in a crude vesicle carrier fraction (CVCF) enriched in transcytotic carriers; (2) slow delivery of [35S]-labeled SC to bile; and (3) paracellular reflux of SC from bile to blood. In conclusion, these data indicate that the secretory and transcytotic pathways remain polarized in cholestatic hepatocytes and suggest that the pIgA-R traffics through postendosomal rab3D-, rab11a-, and syntaxin 2-associated compartments, implicating these proteins in the regulation of transcytosis.

Animals↗

PubMed2005

Structural optimization of pep7, a small peptide extracted from epimorphin, for effective induction of hair follicle anagen.

Epimorphin is representative of a unique class of stromal membrane-anchored proteins that plays distinct functions depending on its membrane topology. When exposed extracellularly, this molecule acts as a morphoregulator for various tissues including hair follicle epithelia. Previous study identified its functional domain (the pep7 domain: SIEQSCDQDE) for hair follicular morphogenesis followed by the successful generation of a chemically modified active peptide. Here, we report optimization of this peptide by the introduction of sequential mutations and subsequent structural determination. We found that three residues from the C-terminus are dispensable, and alternation of the seventh amino acid to an Alanine residue enhanced activity. To favour the biologically active conformation, epsilon-Acp (NH(CH(2))(5)CO) linked to a Cysteine residue was connected at the N-terminus followed by the introduction of an intramolecular disulphide bridge, the modification process of which could be included in the peptide synthesis. The obtained modified peptide, termed 'EPM (epimorphin-derived) peptide', has a Mw of 950 Da and exerts an inductive effect on hair follicle regeneration at a concentration of approximately 0.00001% or even lower. The action of this EPM peptide was more apparent in mice treated with 1% minoxidil, suggesting its potential clinical benefit as a new type of hair-regenerating agent.

Alopecia↗

PubMed2006

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells.

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.

Amiloride↗

PubMed1998

Mint 3: a ubiquitous mint isoform that does not bind to munc18-1 or -2.

Mint 1 and 2 are proteins that bind to munc18-1, an essential component of the synaptic vesicle fusion machinery, and are detectably expressed only in neurons [Okamoto and Südhof, J. Biol. Chem. 272, 31459-31464 (1997)]. Mint 1 and 2 are composed of a variable N-terminal region that includes a conserved munc18-1-binding site, and a constant C-terminal region that contains one PTB and two PDZ domains. We have now identified a third mint isoform, mint 3. Similar to mint 1 and 2, the C-terminal half of mint 3 is composed of one PTB domain and two PDZ domains. However, in contrast to mint 1 and 2, mint 3 lacks an N-terminal munc18-binding domain and does not interact with munc18-1 in yeast two-hybrid assays. Mint 3 is ubiquitously expressed in all tissues, with lowest levels in brain and testis whereas mint 1 and 2 appear to be brain-specific. Our data suggest that mints form a diverse family of proteins with specialized neuronal and ubiquitous isoforms.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Developing rat pineal cells manifest potential of neuronal differentiation in vitro.

The pineal gland in mammals is an endocrine organ and generally does not exhibit neuronal characteristics. However, it is known that under culture conditions, cells from newborn rat pineals express properties characteristic of photoreceptors. Here, we studied the potential of rat pineal cells to differentiate into neuronal cell types using different neural markers. Three phenotype markers characteristic of nerve cells, i.e., intense GABA, neuron-specific antigen (HPC-1) and microtubule-associated protein 2 (MAP2) immunoreactivities, were detected in the pineal culture of newborn rats. Expression of the respective neuronal phenotypes appears to be controlled by different mechanisms; in the normal culture medium containing 5.4 mM KCl, numerous cells were stained intensely with anti-GABA antiserum, whereas only a few were stained intensely either with HPC-1 or MAP2 antibody. In a culture medium with a high concentration of KCl (35 mM), which may induce depolarization of nerve cells, numerous cells became strongly positive for HPC-1 or MAP2; both the cell bodies and the neuritic fibers were stained positively. Since cells intensely immunoreactive to GABA, HPC-1 or MAP2 were not found in intact pineals of the rat, the present results indicate that the neuronal potency of the rat pineal cells is expressed only in vitro and is suppressed in vivo, and that the potency is lost during postnatal development. Norepinephrine at 1 microM, which suppresses differentiation of rhodopsin immunoreactive cells, was ineffective in inducing phenotypic expression of neuronal properties in the present system, indicating that the mechanism of suppression of neuronal properties in the intact pineal may be different from the one for photoreceptors.

Animals↗

PubMed1999

Inhibition of microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein.

HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE. The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane. When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons. These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neurotransmitter release. Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system. Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-deltaTM), which was overexpressed in E. coli. Affinity column chromatography showed that tubulin was found to bind HPC-1 directly. Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule. Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization. Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1. After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.

Amino Acid Sequence↗

PubMed2004

Abl interactor 1 (Abi-1) wave-binding and SNARE domains regulate its nucleocytoplasmic shuttling, lamellipodium localization, and wave-1 levels.

The Abl interactor 1 (Abi-1) protein has been implicated in the regulation of actin dynamics and localizes to the tips of lamellipodia and filopodia. Here, we show that Abi-1 binds the actin nucleator protein Wave-1 through an amino-terminal Wave-binding (WAB) domain and that disruption of the Abi-1-Wave-1 interaction prevents Abi-1 from reaching the tip of the lamellipodium. Abi-1 binds to the Wave homology domain of Wave-1, a region that is required for translocation of Wave-1 to the lamellipodium. Mouse embryo fibroblasts that lack one allele of Abi-1 and are homozygous null for the related Abi-2 protein exhibit decreased Wave-1 protein levels. This phenotype is rescued by Abi-1 proteins that retain Wave-1 binding but not by Abi-1 mutants that cannot bind to Wave-1. Moreover, we uncovered an overlapping SNARE domain in the amino terminus of Abi-1 that interacts with Syntaxin-1, a SNARE family member. Further, we demonstrated that Abi-1 shuttles in and out of the nucleus in a leptomycin B (LMB)-dependent manner and that complete nuclear translocation of Abi-1 in the absence of LMB requires the combined inactivation of the SNARE, WAB, and SH3 domains of Abi-1. Thus, Abi-1 undergoes nucleocytoplasmic shuttling and functions at the leading edge to regulate Wave-1 localization and protein levels.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Characterization of a transformed rat retinal ganglion cell line.

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Animals↗

PubMed1996

Localization of HPC-1/syntaxin 1 in developing rat cerebellar cortex.

In adult rat cerebellum, HPC-1/syntaxin 1 is detected at high density on the plasma membrane of the non-synaptic region of parallel fibers in addition to the synaptic terminal membranes and the synaptic vesicles (Koh, S., Yamamoto, A., Inoue, A., Inoue, Y., Akagawa, Y., Kawamura, Y., Kawamoto, Y., and Tashiro, Y. (1993). J. Neurocytology 22: 995-1005). To assess the possibility that HPC-1/syntaxin 1 participates in the morphogenesis of the nervous system, we examined changes in the localization of HPC-1/syntaxin 1 during postnatal development of the molecular layer of the rat cerebellum. HPC-1/syntaxin 1 appeared in the granule cells in the outer granule cell layer in 3-days-old rat cerebellum when the formation of synapses and the appearance of a synaptic vesicle protein, synaptophysin, had not yet been observed in the molecular layer. At this stage, the granule cells began to form parallel fibers. Confocal laser microscopy and immuno-electron microscopy showed that HPC-1/syntaxin 1 was localized on the extruding plasma membrane of the granule cells to form parallel fibers. In 8-days-old rats, synapses formed between the parallel fibers and the developing dendrites of Purkinje cells, and the HPC-1 immunoreactivity appeared on the axons of parallel fibers and on the synapses. In 21-days-old rats, the HPC-1/syntaxin is involved in the formation of the molecular layer, especially in the axonal growth of the parallel fibers.

Animals↗

PubMed2006

Syntenin-1 is a new component of tetraspanin-enriched microdomains: mechanisms and consequences of the interaction of syntenin-1 with CD63.

Tetraspanins are clustered in specific microdomains (named tetraspanin-enriched microdomains, or TERM) in the plasma membrane and regulate the functions of associated transmembrane receptors, including integrins and receptor tyrosine kinases. We have identified syntenin-1, a PDZ domain-containing protein, as a new component of TERM and show that syntenin-1 specifically interacts with the tetraspanin CD63. Detailed biochemical and heteronuclear magnetic resonance spectroscopy (NMR) studies have demonstrated that the interaction is mediated by the C-terminal cytoplasmic region of the tetraspanin and the PDZ domains of syntenin-1. Upon interaction, NMR chemical shift perturbations were predominantly localized to residues around the binding pocket of PDZ1, indicating a specific mode of recognition of the cytoplasmic tail of CD63. In addition, the C terminus of syntenin-1 has a stabilizing role in the CD63-syntenin-1 association, as deletion of the last 17 amino acids abolished the interaction. The CD63-syntenin-1 complex is abundant on the plasma membrane, and the elevated expression of the wild-type syntenin-1 slows down constitutive internalization of the tetraspanin. Furthermore, internalization of CD63 was completely blocked in cells expressing a syntenin-1 mutant lacking the first 100 amino acids. Previous results have shown that CD63 is internalized via AP-2-dependent mechanisms. Hence, our data indicate that syntenin-1 can counteract the AP-2-dependent internalization and identify this tandem PDZ protein as a new regulator of endocytosis.

Animals↗