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R H Scheller

Publications and source records attributed to R H Scheller.

At least 127 records · Page 7Linked to original sources
PubMed1994

Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.

Agrin is an extracellular matrix component which promotes the clustering of nicotinic acetylcholine receptors (nAChRs) and other proteins at the neuromuscular junction. This aggregation process is one of the earliest steps in synapse formation. Expression of highly active isoforms of agrin, generated by alternative splicing, is restricted to neurons in the central nervous system (CNS) including motoneurons. In the experiments reported here we investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, we expressed truncated soluble forms of the agrin protein in mammalian cells and assessed their clustering activity. Second, we generated a panel of monoclonal antibodies (mAbs) against agrin and mapped their epitopes. Several mAbs block agrin-induced aggregation of nAChRs. One of the mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isoforms. Mapping of the Agr86 epitope suggests that alternative splicing results in a distributed conformational change in the agrin protein. Taken together our data suggest that four domains in the C-terminal 55 kDa of agrin contribute to its nAChR clustering activity.

Agrin↗
PubMed1994

A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering.

Synapse formation is characterized by the accumulation of molecules at the site of contact between pre- and postsynaptic cells. Agrin, a protein implicated in the regulation of this process, causes the clustering of acetylcholine receptors (AChRs). Here we characterize an agrin-binding site on the surface of muscle cells, show that this site corresponds to alpha-dystroglycan, and present evidence that alpha-dystroglycan is functionally related to agrin activity. Furthermore, we demonstrate that alpha-dystroglycan and adhalin, components of the dystrophin-associated glycoprotein complex, as well as utrophin, colocalize with agrin-induced AChR clusters. Thus, agrin may function by initiating or stabilizing a synapse-specific membrane cytoskeleton that in turn serves as a scaffold upon which synaptic molecules are concentrated.

Agrin↗
PubMed1994

Existence of nitric oxide synthase in rat hippocampal pyramidal cells.

It has been proposed that nitric oxide (NO) serves as a key retrograde messenger during long-term potentiation at hippocampal synapses, linking induction of long-term potentiation in postsynaptic CA1 pyramidal cells to expression of long-term potentiation in presynaptic nerve terminals. However, nitric oxide synthase (NOS), the proposed NO-generating enzyme, has not yet been detected in the appropriate postsynaptic cells. We here demonstrate specific NOS immunoreactivity in the CA1 region of hippocampal sections by using an antibody specific for NOS type I and relatively gentle methods of fixation. NOS immunoreactivity was found in dendrites and cell bodies of CA1 pyramidal neurons. Cultured hippocampal pyramidal cells also displayed specific immunostaining. Control experiments showed no staining with preimmune serum or immune serum that was blocked with purified NOS. These results demonstrate that CA1 pyramidal cells contain NOS, as required were NO involved in retrograde signaling during hippocampal synaptic plasticity.

Amino Acid Oxidoreductases↗
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↗
PubMed1994

n-Sec1: a neural-specific syntaxin-binding protein.

We have identified n-Sec1, a rat brain homolog of the yeast Sec1p protein that participates in the constitutive secretory pathway between the Golgi apparatus and the plasma membrane. The rat brain cDNA is predicted to encode a 68-kDa protein with 65% amino acid identity to Drosophila rop, 59% identity to Caenorhabditis elegans unc-18, and 27% identity to Saccharomyces cerevisiae Sec1p. By RNA blot analysis, n-Sec1 mRNA expression is neural-specific. An anti-peptide antiserum directed against the n-Sec1 carboxyl terminus detects a 68-kDa protein in rat brain cytosol and membranes, but not in peripheral tissues. In the presence of syntaxin 1a, a plasma membrane protein implicated in synaptic vesicle docking, n-Sec1 becomes membrane-associated. n-Sec1 binds to syntaxin 1a, 2, and 3 fusion proteins coupled to agarose beads, but not to syntaxin 4 fusion protein or beads coupled to a variety of other proteins. These findings indicate that n-Sec1 is a neural-specific, syntaxin-binding protein that may participate in the regulation of synaptic vesicle docking and fusion.

Amino Acid Sequence↗
PubMed1994

Proteolytic processing of the Aplysia A peptide precursor in AtT-20 cells.

When the Aplysia ELH precursor is expressed in AtT-20 cells, the carboxyterminal derived peptides are packaged and stored in secretory vesicles, while the aminoterminal region of the precursor is constitutively secreted. In contrast, when the highly homologous A peptide precursor is transfected into AtT-20 cells, both aminoterminal and carboxyterminal derived peptides are packaged in storage granules. We propose that this is due to the fact that the initial cleavage of the A peptide precursor occurs more slowly, and perhaps later in the secretory pathway, than the ELH precursor. We further suggest that in the A peptide precursor, the first cleavage occurs after the sorting site resulting in co-packaging of the multiple products derived from a single precursor protein. To determine the structural features of the prohormones responsible for this differential sorting, we made chimeric precursors and determined the rates of the initial cleavage as well as the efficiency of storing the peptide products. From these studies, we conclude that the differential sorting is regulated both by the amino acid sequence of the first processing site, and by more global aspects of the precursor structure.

Animals↗
PubMed1994

The function and differential sorting of a family of aplysia prohormone processing enzymes.

We have cloned four members of the family of subtilisin-like endoproteases expressed in the bag cell neurons of Aplysia and have demonstrated that two of these enzymes are capable of correctly cleaving the egg-laying hormone precursor. The egg-laying hormone precursor undergoes an ordered series of cleavages, such that different peptides are differentially sorted into distinct secretory vesicles. We have used electron microscopic chemistry to demonstrate that at least one processing enzyme is differentially segregated into a class of secretory vesicles containing the bag cell peptides. The segregation of specific endoproteases, along with specific neuropeptides within a given cell type, may ensure appropriate cleavage and prevent inappropriate cleavage of the polyprotein precursors.

Amino Acid Sequence↗
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

A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.

The SNARE hypothesis holds that a transport vesicle chooses its target for fusion when a soluble NSF attachment protein (SNAP) receptor on the vesicle (v-SNARE) pairs with its cognate t-SNARE at the target membrane. Three synaptosomal membrane proteins have previously been identified: syntaxin, SNAP-25 (t-SNAREs), and vesicle-associated membrane protein (VAMP) (v-SNARE); all assemble with SNAPs and NSF into 20S fusion particles. We now report that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin. Synaptotagmin is displaced by alpha-SNAP, suggesting that these two proteins share binding sites on the SNARE complex and implying that synaptotagmin operates as a "clamp" to prevent fusion from proceeding in the absence of a signal. The alpha-SNAP-SNARE complex can bind NSF, and NSF-dependent hydrolysis of ATP dissociates the complex, separating syntaxin, SNAP-25, and VAMP. ATP hydrolysis by NSF may provide motion to initiate bilayer fusion.

Adenosine Triphosphatases↗
PubMed1993

Optimizing fluorescence detection in chemical separations for analyte bands traveling at different velocities.

In many separation techniques, such as field flow fractionation, liquid chromatography, and electrophoresis, chemical species form bands that migrate at distinct velocities. If these bands are to be quantified on-line using a shot-noise-limited detection system, then attention must be given to the data-digitization rate and to the removal rate of molecules from the analyte pool as a result of the detection process. A theory is developed for calculating the signal-to-noise ratio under such conditions, and it is specialized to the case of fluorescence detection in capillary electrophoresis. Using standard detection procedures in which the data-digitization rate and excitation intensity remain constant for the duration of a separation, detection sensitivity can vary by more than a factor of five for bands that arrive at the detection zone between migration times tau fast and 10 tau fast, where tau fast is the time after the start of the separation that the fastest migrating band arrives at the detection zone. To compensate for different band velocities, both the data-digitization rate and the excitation intensity must be decreased as separation time (tau) increases by the factor tau fast/tau. Only when these corrections are made can uniform sensitivity with the highest possible signal-to-noise ratio be achieved for each peak. These predictions are experimentally tested and compare favorably to observations.

Electrophoresis↗
PubMed1993

Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells.

VAMP (synaptobrevin) is a highly conserved membrane protein originally described as a component of brain synaptic vesicles. The Drosophila melanogaster VAMP-encoding gene (syb) comprises five exons. Splicing exons 1,2,3,4,5 (syb-b) results in a protein with a C-terminal hydrophobic domain and a negligible intraluminal domain. Splicing exons 1,2,3,5 (syb-a) predicts a protein with a 20-amino-acid luminal domain at the C terminus. The ratio of syb-a to syb-b transcripts is highly regulated during development. The syb transcripts show no enrichment in the nervous system and are present in very early embryos, well before neurogenesis. The greatest concentration of syb transcripts was found in cells of the gut and malpighian tubules. Thus, syb may have a general role in membrane trafficking and, perhaps, a role in the secretion of digestive enzymes.

Alternative Splicing↗