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PubMed2001

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. I. Mitogen-activated protein kinase cascade.

Alterations in the phosphorylation state of the microtubule-associated protein tau have been associated with the pathogenesis of neurofibrillary degeneration as well as with a neuroprotective action against apoptotic cell death. Mitogen-activated protein kinases (MAPK) phosphorylate tau protein in vitro but the pathophysiological significance of this tau phosphorylation and its effects on neuronal viability is far from clear. Moreover, an in vivo model of activation of MAPK, a key candidate for in vivo tau phosphorylation, is still lacking. The aim of the present study and the accompanying paper was to establish an animal model of stimulated MAPK and to analyse the consequences on tau phosphorylation and the neuronal cytoskeleton. We took advantage of transgenic mice with neurone-specific expression of activated ras protein (p21H-ras(Val12)). The expression of the transgene in these animals is forced to a subset of neurones by the use of the synapsin I promoter. Activity of B-raf was elevated by 37%, while activity of MAPK (ERK1/ERK2) was increased by 25% associated with a subcellular redistribution from the cytoplasmic to the nuclear compartment. Kinases downstream of MAPK such as p90rsk and glycogen synthase kinase 3beta were only marginally affected. Activity of p70S6 kinase was unaltered. The present model might be useful to study the effects of activation of the MAPK cascade on tau phosphorylation and its cell biological sequelae.

Animals↗
PubMed2001

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. II. Cytoskeletal proteins and dendritic morphology.

In the present study, we analysed changes in the expression, subcellular distribution and phosphorylation state of the microtubule-associated protein tau and other cytoskeletal proteins after neurone-specific activation of the mitogen-activated protein kinase (MAPK) in the CNS in vivo. We used transgenic mice with a neurone-specific expression of activated ras protein (p21H-ras(Val12), synapsin I promoter) that is associated with an augmented activity of the MAPK. Chronic activation of MAPK cascade influenced tau protein phosphorylation, localisation and dendritic morphology. While the amount of tau protein was elevated by 9%, phospho-epitopes detected by the monoclonal antibodies AT270, 12E8 and SMI34 were increased by about 21%, 40% and 59% respectively. Steady-state levels of tau mRNA were not affected. Thus, the increase in tau protein was most likely due to stabilisation of tau protein by augmented phosphorylation. While in wild-type animals tau protein was preferentially localised in axons, a prominent immunoreactivity was found in the somatodendritic compartment of transgenic mice. This subcellular translocation typically seen in pyramidal neurones was associated with an increase in the dendritic calibre by about 30% and is paralleled by an increase in tubulin of 19%. We were unable to obtain any morphological indication of neurodegenerative processes in these animals. We suggest that the moderate increase in tau protein and phosphorylation may be part of the neuroprotective mechanism. However, further studies on aged transgenic mice will be necessary to establish potential effects on neuronal viability.

Animals↗
PubMed2003

Heterogeneity of endocytic proteins: distribution of clathrin adaptor proteins in neurons and glia.

Clathrin adaptor protein (AP)180 is a synaptic protein that regulates the assembly of clathrin-coated vesicles. Several endocytic proteins including AP2, CALM, and epsin 1 have functions or molecular structures similar to AP180. We determined if AP180 associates with functional synapses in cultured hippocampal neurons. We also compared the expression pattern of AP180 with the other endocytic proteins. The distribution of AP180 corresponded with the synaptic vesicle-associated protein synapsin I, and with functional presynaptic terminals labeled with the styryl dye FM1-43. Synaptic AP2 colocalized with AP180, but the distribution of AP2 was not limited to synapses of neurons and it was also expressed in glia. CLAM and epsin 1 immunoreactivities were also detected in both neurons and glia. Unlike AP180, the neuronal immunoreactivity of CALM was not intense in the synaptic puncta. Epsin 1 immunoreactivity was found in both synaptic and extrasynaptic sites, and its synaptic distribution only partially overlapped with that of AP180. These results support roles for AP180 in synaptic function in neurons. The findings also provide information on the distribution of AP2, CALM, and epsin 1 in cells of the nervous system that suggest different roles for these endocytic proteins in the biology of these cells.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Colchicine administration in the rat central nervous system induces SNAP-25 expression.

he arrest of axonal transport by colchicine administration has been extensively used in immunocytochemical studies to increase the levels of neuroactive compounds in neuronal somata. In order to study the accumulation rates of a variety of proteins with location and physiological action at the synaptic terminal, we analysed, by immunocytochemical methods, the neuronal cell body content of these synaptic proteins in colchicine-injected rats. In sham-injected animals, all synaptic proteins tested were essentially observed in nerve fibres and terminal boutons. After colchicine administration, intense SNAP-25 immunoreactivity was found in many neuronal cell bodies throughout the CNS. In contrast, immunostaining for the rest of the synaptic proteins analysed (syntaxin 1A and 1B, synaptobrevin I and II, Rab3A, synaptophysin, synapsin I, synaptotagmin I and GAP-43) was virtually absent in neuronal cell bodies in treated animals. Furthermore, northern blot and in situ hybridization analysis revealed an increase in SNAP-25a and SNAP-25b messenger RNA isoforms in the brains of adult colchicine-administered animals. In addition, colchicine administration in five-day-old rat pups induced a notable increase in both SNAP-25 transcript isoforms. The present results indicate that in vivo colchicine administration, under conditions known to inhibit axoplasmic transport, upregulates SNAP-25 expression in the rat brain.

Animals↗
PubMed1999

Endocytic vacuoles formed following a short pulse of K+ -stimulation contain a plethora of presynaptic membrane proteins.

It is now well established that the membrane of synaptic vesicles is recycled following exocytosis. However, little is known concerning the identity of the primary or secondary endocytic structures and their molecular composition. Using cultured rat cerebellar granule cells we combined uptake of horseradish peroxidase as a fluid phase marker and immunogold labeling for a variety of presynaptic proteins to assess the molecular identity of the stimulation-induced endocytic compartments. Short periods (5 or 30 s) of stimulation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vacuoles in the axonal varicosities as the apparent primary endocytic compartment. Horseradish peroxidase-filled synaptic vesicles were formed when stimulated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contained the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and in addition SNAP-25. No incorporation of vesicle proteins into the plasma membrane was observed. Horseradish peroxidase-filled vesicles and vacuoles generated on incubation of unstimulated granule cells with horseradish peroxidase for prolonged periods of time were equally immunolabeled. Renewed stimulation of prestimulated granule cells with either 100 mM KCl or 30 microM Ca2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-filled vacuoles suggesting that the vacuolar membrane compartment was exocytosis-competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synaptic vesicle membrane compartment. The primary endocytic compartment represents not a synaptic vesicle but a somewhat larger vesicle protein-containing vacuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membrane and membrane-associated proteins and presumably also major functional properties.

Animals↗
PubMed2000

The alpha7-nicotinic acetylcholine receptor and the pathology of hippocampal interneurons in schizophrenia.

This paper is a review of a recent findings on the pathology of hippocampal interneurons in schizophrenia, with specific emphasis on a protein expressed by these cells, the alpha7-nicotinic acetylcholine receptor subunit. Convergent information indicates that interneurons in the hippocampus and other forebrain structures are decreased in number and function in subjects with schizophrenia. Among the neurochemical markers that are decreased in the hippocampus are synapsin I, cholecystokinin, somatostatin, glutamic acid decarboxylase, and nitric oxide synthase. GABA uptake sites and the GABA synthetic enzyme glutamic acid decarboxylase are also diminished. Included among these findings is decreased binding of alpha-bungarotoxin, which binds to low-affinity nicotinic acetylcholine receptors, such as the alpha7-nicotinic receptor. Co-labeling experiments in rodents indicate that these markers are expressed on overlapping populations of hippocampal interneurons. Thus, the finding of decreased neurochemical function of hippocampal interneurons is a widely replicated finding, with different groups reporting markedly similar findings using independent post mortem samples and different neurochemical strategies. Decreased alpha-bungarotoxin binding or decreased alpha7-nicotinic receptor immunoreactivity has also been found in the frontal cortex and in the nucleus reticularis thalami of schizophrenic subjects. The alpha7-nicotinic receptor subunit gene on chromosome 15q14 is a site of heritability for schizophrenia and bipolar affective disorder, and in, particular, for a deficit in inhibitory neuronal function associated with these illnesses. Thus, the post mortem data are further supported by psychophysiologic and genetic investigations that indicate a deficit in inhibitory interneuronal function, involving the alpha7-nicotinic receptor. The alpha7-receptor is a ligand-gated ion channel that admits calcium ions into cells, and it has been proposed to have various developmental roles. Its malfunction may be part of the developmental pathogenesis of schizophrenia.

Hippocampus↗
PubMed2001

Evidence for targeted vesicular glutamate exocytosis in osteoblasts.

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express functional receptors for the excitatory amino acid, glutamate, indicating that a signaling pathway analogous to synaptic neurotransmission exists in bone. Here, we show that osteoblasts also express the essential molecular framework for regulated glutamate exocytosis to occur as is present in presynaptic neurons. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and northern and western blotting is used to show expression of the target membrane-SNARE (soluble NSF attachment protein receptor), proteins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobrevin), the minimum molecular requirements for core exocytotic complex formation. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocalizing with immunoreactive glutamate and the synaptic vesicle-specific protein, synapsin I. We also identify multiple accessory proteins associated with vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synaptophysin, which have varied roles in regulated glutamate exocytosis. mRNA for the putative Ca(2+)-dependent regulators of vesicle recycling activity, synaptotagmin I (specialized for fast Ca(2+)-dependent exocytosis as seen in synaptic neurotransmission), and the GTP-binding protein Rab3A are also identified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. These data provide compelling evidence that osteoblasts are able to direct glutamate release by regulated vesicular exocytosis, mimicking presynaptic glutamatergic neurons, showing that a process with striking similarity to synaptic neurotransmission occurs in bone.

Animals↗
PubMed2003

Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides.

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphoproteomics applications). Quantitation of the side reaction products using three synthetic unphosphorylated peptides showed varying conversion efficiencies; at maximum, 1.7% of unphosphorylated peptide was converted to the affinity-tagged form. Inclusion of EDTA into the reaction reduced the side reaction but also greatly reduced the conversion efficiency of one of the phosphoserine residues of ovalbumin, suggesting a role for trace metal ions in the beta-elimination chemistry. Despite the presence of the side reaction, the affinity strategy was shown to be effective at enriching phosphopeptides from fairly complex peptide mixtures. The strategy was applied to the analysis of in vitro phosphorylation of bovine synapsin I by Ca(2+)/calmodulin-dependent kinase II, resulting in the identification of four phosphorylation sites, two of which have not been previously reported.

Animals↗
PubMed1990

Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase.

Previously, it was reported that smooth muscle caldesmon is a protein kinase and is autophosphorylated [Scott-Woo, G.C., & Walsh, M.P. (1988) Biochem. J. 252, 463-472]. We separated a Ca2+/calmodulin-dependent protein kinase from caldesmon in the presence of 15 mM MgCl2. The Ca2+/calmodulin-dependent caldesmon kinase was purified by using a series of liquid chromatography steps and was characterized. The subunit molecular weight (MW) of the kinase was 56K by SDS gel electrophoresis and was autophosphorylated. After the autophosphorylation, the kinase became active even in the absence of Ca2+/calmodulin. The substrate specificity of caldesmon kinase was similar to the rat brain calmodulin-dependent multifunctional protein kinase II (CaM PK-II) and phosphorylated brain synapsin and smooth muscle 20-kDa myosin light chain. The purified kinase bound to caldesmon, and the binding was abolished in the presence of high MgCl2. Enzymological parameters were measured for smooth muscle caldesmon kinase, and these were KCaM = 32 nM, KATP = 12 microM, Kcaldesmon = 4.9 microM, and KMg2+ = 1.1 mM. Optimum pH was 7.5-9.5. The observed properties were similar to brain CaM PK-II, and, therefore, it was concluded that smooth muscle caldesmon kinase is the isozyme of CaM PK-II in smooth muscle.

Animals↗
PubMed2003

Catalytic and regulatory domains of doublecortin kinase-1.

Doublecortin kinase-1 (DCK1) is a newly described multidomain protein kinase with a sequence significantly similar to those of both CaM kinases (CaMKs) and doublecortin, the product of the gene mutated in X-linked lissencephaly/double cortex syndrome, a severe developmental disorder of the nervous system. Functional studies have revealed microtubule binding and polymerization activities of the doublecortin domain, yet little is known regarding the enzymatic properties and regulation of the kinase catalytic domain. We have identified and report here notable similarities as well as differences between the catalytic and regulatory properties of DCK1 and those of the CaMKs. Using synthetic peptide substrates modeled on synapsin I, a substrate recognition motif for DCK1 of Hyd-Arg-Arg-X-X-Ser/Thr-Hyd was derived. The similarity of this motif to that of CaMKI [Lee, J. C., Kwon, Y.-G., Lawrence, D. S., and Edelman, A. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6413-6417] is consistent with the 59% level of amino acid sequence similarity between their catalytic domains. DCK1 catalytic activity is enhanced by mutagenic introduction of negative charge at Thr-239, a residue in a position equivalent to that of Thr-177 of CaMKI, the activation loop site for regulation by CaM kinase kinase. Unlike CaMKs, DCK1 is not directly activated by Ca(2+)-bound CaM. However, truncation of a pseudosubstrate-like sequence in the C-terminus of DCK1 results in an approximately 6-fold enhancement of activity. Thus, DCK1 demonstrates the potential to be regulated by relief of autoinhibition in response to signal(s) distinct from Ca(2+)-bound CaM and potentially by activation loop phosphorylation and to phosphorylate intracellular targets at sites similar to those recognized by CaMK pathways.

Animals↗
PubMed1998

X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli.

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second step of the de novo purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. GAR-syn containing an N-terminal polyhistidine tag was expressed as the SeMet incorporated protein for crystallographic studies. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multiple-wavelength anomalous diffraction (MAD) phase determination and refined to 1.6 A resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas the smaller B domain is extended outward. Two hinge regions, which might readily facilitate interdomain movement, connect the B domain and the main core. A search of structural databases showed that the structure of GAR-syn is similar to D-alanine:D-alanine ligase, biotin carboxylase, and glutathione synthetase, despite low sequence similarity. These four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-binding enzyme with low sequence similarity but unknown function, synapsin Ia, was also found to share high structural similarity with GAR-syn. Interestingly, the GAR-syn N domain shows similarity to the N-terminal region of glycinamide ribonucleotide transformylase and several dinucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of these models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP binding belong to the A or B domains. The N and C domains appear to be largely responsible for substrate specificity. The structure of GAR-syn allows modeling studies of possible channeling complexes with PPRP amidotransferase.

Adenosine Triphosphate↗
PubMedDate not supplied

Phosphoproteomic analysis of synaptosomes from human cerebral cortex.

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.

Amino Acid Sequence↗
PubMed2003

Thyroid hormone enhances the formation of synapses between cultured neurons of rat cerebral cortex.

1. Thyroid hormones play important roles in the development of the brain. Increasing evidence suggests that the deprivation of thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive. In this study, we investigated the effects of thyroid hormones on synapse formation between cultured rat cortical neurons, using a system to estimate functional synapse formation in vitro. 2. Exposure to 10(-9) M thyroid hormones, 3,5,3'-triiodothyronine or thyroxine, caused an increase in the frequency of spontaneous synchronous oscillatory changes in intracellular calcium concentration, which correlated with the number of synapses formed. 3. The detection of synaptic vesicle-associated protein synapsin I by immunocytochemical and immunoblot analysis also confirmed that exposure to thyroxine facilitated synapse formation. 4. The presence of amiodarone, an inhibitor of 5'-deiodinase, or amitrole, a herbicide, inhibited the synapse formation in the presence of thyroxine. 5. In conclusion, we established a useful in vitro assay system for screening of miscellaneous chemicals that might interfere with synapse formation in the developing CNS by disrupting the thyroid system.

Animals↗
PubMed2004

Endogenous dopamine maintains synchronous oscillation of intracellular calcium in primary cultured-mouse midbrain neurons.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

Action Potentials↗
PubMed1992

Actin-dependent organelle movement in squid axoplasm.

Studies of organelle movement in axoplasm extruded from the squid giant axon have led to the basic discoveries of microtubule-dependent organelle motility and the characterization of the microtubule-based motor proteins kinesin and cytoplasmic dynein. Rapid organelle movement in higher animal cells, especially in neurons, is considered to be microtubule-based. The role of actin filaments, which are also abundant in axonal cytoplasm, has remained unclear. The inhibition of organelle movement in axoplasm by actin-binding proteins such as DNase I, gelsolin and synapsin I has been attributed to their ability to disorganize the microtubule domains where most of the actin-filaments are located. Here we provide evidence of a new type of organelle movement in squid axoplasm which is independent of both microtubules and microtubule-based motors. This movement is ATP-dependent, unidirectional, actin-dependent, and probably generated by a myosin-like motor. These results demonstrate that an actomyosin-like mechanism can be directly involved in the generation of rapid organelle transport in nerve cells.

Actin Cytoskeleton↗
PubMed1986

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.

Animals↗
PubMed1994

Expression of vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin II and cellubrevin in rat skeletal muscle and in a muscle cell line.

Molecular studies have identified a family of synaptic vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins) which have been implicated in synaptic vesicle docking and/or fusion with plasma membrane proteins. Here we demonstrate the expression of two members of this family, VAMP-2/synaptobrevin II and cellubrevin, in skeletal muscle, a tissue with both constitutive and regulated membrane traffic. The 18 kDa VAMP-2 polypeptide was detected in purified membrane fractions from adult skeletal muscle and from L6 myotubes in culture, demonstrating that the presence of this protein in the isolated muscle membrane fractions is not the result of contamination by ancillary tissues such as peripheral nerve. Furthermore, skeletal muscle and the muscle cell line also expressed cellubrevin, a VAMP-2 homologue of 17 kDa; which is much less abundant in brain cells. Both VAMP-2 and cellubrevin were preferentially isolated in membrane fractions rich in plasma membranes, and were less concentrated in light microsomes and other internal membrane fractions of mature muscle or muscle cells in culture. Interestingly, both VAMP-2 and cellubrevin were much more abundant in the differentiated L6 myotubes than in their precursor myoblasts, suggesting that they are required for functions of differentiated muscle cells. The identity of both polypeptides was further confirmed by their susceptibility to proteolysis by Clostridium tetanus toxin. Expression of these products was further established by the presence of mRNA transcripts of VAMP-2 and cellubrevin, but not of VAMP-1, in both skeletal muscle and L6 myotubes. In contrast, other synaptic vesicle and docking/fusion components were undetectable, such as VAMP-1, SNAP25 and syntaxin 1A/1B, as were synaptophysin and synapsin Ia/Ib, proteins which are believed to be involved in sensing the signal for neuronal exocytosis. It is concluded that VAMP-2 and cellubrevin are expressed in skeletal muscle cells and may each participate in specific processes of intracellular membrane traffic.

Amino Acid Sequence↗
PubMed2002

Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene.

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.

5' Untranslated Regions↗