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R H Scheller

Publications and source records attributed to R H Scheller.

At least 37 records · Page 2Linked to original sources
PubMed1994

Specificity and regulation of a synaptic vesicle docking complex.

Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.

Amino Acid Sequence↗
PubMed1994

Mechanisms of vesicle docking and fusion: insights from the nervous system.

Upon stimulation of nerve cells, synaptic vesicles fuse with the presynaptic plasma membrane to release neurotransmitter. The biochemical pathway responsible for synaptic-vesicle docking and fusion is now being elucidated. Many of the proteins implicated in this process have homologs elsewhere in the cell. The docking and fusion mechanism discussed in this review may account for the specificity of vesicular trafficking throughout both regulated and constitutive secretory pathways.

Animals↗
PubMed1994

Molecular correlates of synaptic vesicle docking and fusion.

The mechanisms responsible for neurotransmitter release at the synapse have been extensively studied using biochemical, genetic, and cell biological approaches. Several significant advances have recently contributed towards an improved understanding of the molecular details of both synaptic vesicle docking and fusion, and of the general process of vesicle-mediated membrane trafficking.

Animals↗
PubMed1994

Secretion in AtT-20 cells stably transfected with soluble synaptotagmins.

Synaptotagmin (p65) is an integral membrane secretory vesicle-specific protein with two cytoplasmic repeats homologous to the C2 regulatory domain of protein kinase C. Synaptotagmin has been implicated in the regulation of neurotransmitter release from nerve growth factor-differentiated PC12 cells and from synapses in Drosophila, Caenorhabditis elegans, and squid. To address the function of synaptotagmin in endocrine cells, fragments of rat synaptotagmin I were stably expressed in the mouse anterior pituitary cell line AtT-20. The logic of these experiments is that the fragments may interfere with the endogenous synaptotagmin machinery, thus producing a dominant-negative phenotype. Transfected cells expressed the expected fragments that were comprised of either the first C2 repeat, the second C2 repeat, or the entire cytoplasmic domain. The fragments were localized to both soluble and membrane-associated cellular fractions, despite the absence of the transmembrane domain. The second C2 repeat was shown to coimmunoprecipitate with endogenous synaptotagmin, suggesting that protein-protein interactions are mediating the membrane association of the fragments. These fragments had no effect on the targeting of regulated secretory vesicles or on regulated secretion as assayed by the release of ACTH and [3H]choline. Constitutive secretion assayed by the release of glycosaminoglycan side chains was also unaffected, as was the endocytic pathway monitored by the uptake and clearance of transferrin. These data suggest either the existence of a redundant pathway in secretion or that regulated membrane traffic in endocrine cells does not require synaptotagmin.

Adenoma↗
PubMed1994

Differential expression of synaptic vesicle protein 2 (SV2) isoforms.

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.

Animals↗
PubMed1993

The syntaxin family of vesicular transport receptors.

Syntaxins A and B are nervous system-specific proteins implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. A family of syntaxin-related proteins from rat has been identified that shares 23%-84% amino acid identity. Each of the six syntaxins terminate with a carboxy-terminal hydrophobic domain that anchors the protein on the cytoplasmic surface of cellular membranes. The syntaxins display a broad tissue distribution and, when expressed in COS cells, are targeted to different subcellular compartments. Microinjection studies suggest that the nervous system-specific syntaxin 1A is important for calcium-regulated secretion from neuro-endocrine PC12 cells. These results indicate that the syntaxins are a family of receptors for intracellular transport vesicles and that each target membrane may be identified by a specific member of the syntaxin family.

Amino Acid Sequence↗
PubMed1993

The molecular machinery for secretion is conserved from yeast to neurons.

A variety of approaches have been utilized to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway. Two approaches that have been particularly fruitful include the genetic dissection of the yeast secretory pathway and the biochemical characterization of proteins involved in the synaptic vesicle membrane trafficking in the mammalian nerve terminal. The recent convergence of these approaches suggests that common mechanisms may underlie a wide variety of vesicle-mediated transport steps. We discuss the results that support this possibility and propose a model for synaptic vesicle docking and fusion that incorporates evolutionarily conserved elements that may be part of a constitutive fusion machinery and specialized elements that may mediate regulatory events that are specific to the process of neurotransmitter release.

Animals↗
PubMed1993

Brain contains two forms of synaptic vesicle protein 2.

Molecular cloning of a cDNA encoding synaptic vesicle protein 2 (SV2) revealed that it is homologous to a family of proton cotransporters from bacteria and fungi and to a related family of glucose transporters found in mammals. The similarity to proton cotransporters raised the possibility that SV2 might mediate the uptake of neurotransmitters into vesicles, an activity known to require a proton gradient. To determine whether SV2 is a member of a family of vesicular proteins, we used the SV2 clone to screen for similar cDNAs in rat brain. We characterized 42 clones, 25 of which encode SV2 and 4 of which encode a protein, SV2B, that is 65% identical and 78% similar to SV2. The protein encoded by SV2B cDNA is recognized by the monoclonal antibody that defines the SV2 protein. When SV2B is expressed in COS cells, antibody labeling is reticular in nature, suggesting that SV2B, like SV2 (hence, SV2A), is segregated to intracellular membranes. The expression of SV2B is limited to neural tissue. While both forms of SV2 are expressed in all brain regions, SV2B is expressed at highest levels in the cortex and hippocampus, whereas the highest level of expression of SV2A is in subcortical regions. Therefore, the SV2 proteins, like other characterized synaptic vesicle proteins, comprise a small gene family.

Amino Acid Sequence↗
PubMed1992

p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C.

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.

Amino Acid Sequence↗
PubMed1992

SV2, a brain synaptic vesicle protein homologous to bacterial transporters.

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein specifically localized to secretory vesicles in neurons and endocrine cells. As a first step toward understanding the function of SV2 in neural secretion, a rat brain complementary DNA (cDNA) that encodes SV2 was isolated and characterized. Analyses of this cDNA predict that SV2 contains 12 transmembrane domains. The NH2-terminal half of the protein shows significant amino acid sequence identity to a family of bacterial proteins that transport sugars, citrate, and drugs. Expression of the SV2 cDNA in COS cells yielded a high level of SV2-like immunoreactivity distributed in a reticular and punctate pattern, which suggests localization to intracellular membranes. Its localization to vesicles, predicted membrane topology, and sequence identity to known transporters suggest that SV2 is a synaptic vesicle-specific transporter.

Amino Acid Sequence↗
PubMed1992

Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.

Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35 proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.

Amino Acid Sequence↗
PubMed1992

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.

Amino Acid Sequence↗
PubMed1992

RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes.

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Agrin↗
PubMed1992

Agrin and the organization of the neuromuscular junction.

Agrin is a component of the synaptic extracellular matrix and may regulate the organization of acetylcholine receptors and other synaptic molecules in both synapse regeneration and development. Analyses of cDNAs encoding agrin define a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and epidermal growth factor repeats.

Agrin↗