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R H Scheller

Publications and source records attributed to R H Scheller.

At least 145 records · Page 8Linked to original sources
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

Identification of a 34 kDa protein specific to synaptic vesicles.

In this study, we used synaptic vesicles purified from the electric organ of marine electric rays to search for novel molecules which have important functions in synaptic transmission. Proteins that copurified with synaptic vesicles were used to immunize rats, and the resulting antisera were then used to further characterize the vesicle proteins. One of the antisera recognizes a protein of 34 kDa, p34, that has several characteristics which suggest it is a synaptic vesicle specific protein: (1) it copurifies exclusively with the synaptic vesicle peak during permeation chromatography on a controlled pore glass beads column, (2) it can be immunoprecipitated with intact synaptic vesicles and (3) it is specifically localized to the nervous system. The results suggest that p34 is a synaptic vesicle specific protein with a widespread distribution in the nervous system.

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↗
PubMed1993

Association of three small GTP-binding proteins with cholinergic synaptic vesicles.

Several small (low molecular weight) GTP-binding proteins are associated with cholinergic synaptic vesicles derived from the electric organ of electric ray. Using GTP overlay techniques and direct micro sequencing we analyzed the association of small GTP-binding proteins with synaptic vesicles. Both experimental procedures revealed the specific occurrence of multiple small GTP-binding proteins with this organelle. Moreover, direct amino acid sequence analysis assigned at least three different small GTP-binding proteins, ora3, o-ral and o-rab3, to the vesicular compartment. Furthermore, the data reflect the relative abundance of these three proteins on the vesicle membrane, thereby demonstrating the predominant occurrence of o-rab3, the only exclusively synaptic vesicle specific small GTP-binding protein.

Amino Acid Sequence↗
PubMed1993

A role for synaptotagmin (p65) in regulated exocytosis.

Proteins that are specifically localized to synaptic vesicles in the nervous system have been proposed to mediate aspects of synaptic transmission. Antibodies raised against the cytoplasmic domains of five of these proteins, vamp, rab3A, synaptophysin, synaptotagmin, and SV2, were used to investigate their function. Microinjection of monoclonal and polyclonal antibodies raised against synaptotagmin (p65), but not the other vesicle proteins, decreases K+/Ca(2+)-mediated dopamine beta-hydroxylase surface staining, a measure of regulated secretion in PC12 cells. Microinjection of a soluble fragment of synaptotagmin encompassing one of the domains homologous to the C2 regulatory region of protein kinase C, but lacking the membrane anchor, also inhibits evoked dopamine beta-hydroxylase surface staining. These results provide support for the hypothesis that synaptotagmin, a Ca(2+)- and phospholipid-binding protein, is important for regulated exocytosis in neurons.

Amino Acid Sequence↗
PubMed1993

Developmental regulation of highly active alternatively spliced forms of agrin.

Agrin is an extracellular matrix protein involved in clustering acetylcholine receptors during development of the neuromuscular junction. We have previously shown that alternative splicing at three sites generates multiple forms of rat agrin and that a novel 8 amino acid insert is the most important in determining biological activity. In the present study we have examined the expression of agrin during development with particular emphasis on determining the tissue distribution of the splicing variants at each site. Our principal observation is that the variants containing the sequence most responsible for biological activity are expressed exclusively in neural tissue and that their expression is highly regulated during development. We also show that muscle expresses less active forms and that agrin immunoreactivity during synaptogenesis is initially not limited to synaptic sites, but becomes progressively restricted to the synapse as development proceeds.

Aging↗
PubMed1993

The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans.

Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the y site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.

Agrin↗
PubMed1993

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.

Amino Acid Sequence↗
PubMed1993

A rab protein regulates the localization of secretory granules in AtT-20 cells.

Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles. Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles. When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH. Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation. The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.

Adrenocorticotropic Hormone↗
PubMed1993

Ceramide 1-phosphate phosphatase activity in brain.

Recent studies have implicated sphingolipids in a variety of intracellular signaling systems. The finding that a calcium-stimulated ceramide kinase copurifies with neurotransmitter-containing vesicles suggests that ceramide, or one of its metabolites, has a role in neurotransmitter release. As a step toward understanding the role of ceramide kinase in vesicle functioning, this study sought to determine the metabolic fate of the product, ceramide 1-phosphate. We report that ceramide 1-phosphate is not deacylated by brain ceramidases to produce sphingosine 1-phosphate. It is, however, the substrate for a phosphatase activity that we name ceramide 1-phosphate phosphatase (CPPase). Subcellular fractionation studies suggest that CPPase is found in the synaptic terminal and is associated with both synaptic vesicle and plasma membranes. Divalent cations, most notably calcium, inhibit CPPase activity although not at concentrations that activate ceramide kinase. The existence of both ceramide kinase and CPPase activities at the synapse suggests that ceramide 1-phosphate production regulates some aspect of synaptic vesicle functioning.

Amidohydrolases↗
PubMed1993

Synaptic vesicle proteins and regulated exocytosis.

The recent identification of novel proteins associated with the membranes of synaptic vesicles has ignited the field of molecular neurobiology to probe the function of these molecules. Evidence is mounting that the vesicle proteins vamp (synaptobrevin), rab3A, synaptophysin, synaptotagmin (p65) and SV2 play an important role in regulated exocytosis, by regulating neurotransmitter uptake, vesicle targeting and fusion with the presynaptic plasma membrane.

Animals↗
PubMed1993

Identification and characterization of Drosophila genes for synaptic vesicle proteins.

Proteins associated with synaptic vesicles are likely to control the release of neurotransmitter. Because synaptic transmission is fundamentally similar between vertebrates and invertebrates, vesicle proteins from vertebrates that are important for synaptic transmission should be present in Drosophila as well. This investigation describes Drosophila homologs of vamp, synaptotagmin, and rab3 that are expressed in a pattern consistent with a function in Drosophila neurotransmission. One previously reported candidate (syb), a Drosophila homolog of the vamp or synaptobrevin proteins, has been shown to be expressed at very low levels in neurons and is most abundant in the gut. A neuronal Drosophila vamp (n-syb) is described here and is localized to chromosome band 62A. Northern analysis and in situ hybridizations to mRNA indicate that the novel vamp, as well as the genes for synaptotagmin (syt) and rab3 (drab3), is expressed in the Drosophila nervous system. These genes are widely (perhaps ubiquitously) expressed in the nervous system and we have no evidence of additional neuronal isoforms of synaptotagmin, vamp, or rab3. Immunoreactivity for synaptotagmin and vamp is located in synaptic regions of the nervous system. This distribution suggests that these molecules are components of synaptic vesicles in Drosophila. The conserved structure and neuronal expression pattern of these genes indicate that they may function in processes that are required for both vertebrate and invertebrate synaptic transmission. Because of their distribution in the nervous system and because n-syb, synaptotagmin, and drab3 do not appear to be in a family of functionally redundant homologs, we predict that mutation of these genes will have a profound neurological phenotype and that they are therefore good candidates for a genetic dissection in Drosophila.

Amino Acid Sequence↗
PubMed1993

Casein kinase II phosphorylates the synaptic vesicle protein p65.

p65 (synaptotagmin), an abundant synaptic vesicle protein, has been implicated in the processes of vesicle docking and fusion. To characterize further the properties of this important neuronal protein, we have investigated its phosphorylation in vitro. Immunoprecipitation of p65 results in coprecipitation of a protein kinase that phosphorylates p65 as well as syntaxin, a plasma membrane protein that interacts with p65. p65 is phosphorylated on a threonine residue (Thr-128) within the cytoplasmic domain near the transmembrane region. The coprecipitating protein kinase was identified as casein kinase II based on its catalytic properties, the sequence surrounding Thr-128, and Western blot analysis of the anti-p65 immunoprecipitates. Affinity chromatography utilizing bacterially expressed fragments of p65 demonstrated that casein kinase II interacts with a domain of p65 distinct from the phosphorylation site. In a synaptic vesicle fraction, the phosphorylation of p65 is stimulated by sphingosine and by detergent solubilization, suggesting that p65 phosphorylation may be subject to regulatory processes.

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↗