rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.
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Amino Acid Sequence↗
Biomedical subjects
R H Scheller
Publications and source records attributed to R H Scheller.
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.
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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↗
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↗
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↗
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↗
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↗
Synaptic vesicle membrane proteins interact to form a multimeric complex.
Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.
Animals↗
Agrin: a synaptic basal lamina protein that regulates development of the neuromuscular junction.
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Agrin↗
Agrin mediates cell contact-induced acetylcholine receptor clustering.
One of the important events in synapse formation is the accumulation of neurotransmitter receptors beneath the presynaptic nerve terminal. Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors when bath-applied to muscle fibers in culture. When a cDNA encoding a putative agrin protein is transfected into cells, the molecule is secreted and concentrated on the extracellular surface. Coculture of transfected cells with muscle fibers induces the formation of receptor patches at contact sites. These results demonstrate that expression of a single gene encoding agrin confers receptor clustering that is restricted to specific sites of cell-muscle contact.
Agrin↗
Peptide processing and targeting in the neuronal secretory pathway.
The abdominal ganglion of the marine mollusk Aplysia contains a pair of identified neuronal clusters, the bag cells, which control egg laying by means of a number of unique regulatory mechanisms. Each neuron in the bag cell clusters synthesizes several peptides derived from a single prohormone and packages them into separate vesicles. These vesicles are then differentially localized in specific neuronal processes, thus segregating peptides destined for autocrine and hormonal release sites. Therefore in this system, protein trafficking through the secretory pathway organizes multiple peptide neurochemical messengers to efficiently regulate simple behaviors.
Animals↗
Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.
A fluorescence detection system for capillary zone electrophoresis is described in which a charged-coupled device (CCD) views a 2-cm section of an axially illuminated capillary column. The CCD is operated in two readout modes: a snapshot mode that acquires a series of images in wavelength and capillary position, and a time-delayed integration mode that allows long exposure times of the moving analyte zones. By use of the latter mode, the ability to differentiate a species based on both its fluorescence emission and migration rate is demonstrated for fluorescein and sulforhodamine 101. The detection limit for fluorescein isothiocyanate (FITC) is 1.2 X 10(-20) mol; detection limits for FITC-amino acids are in the (2-8) X 10(-20) mol range.
Amino Acids↗
A family of ras-like GTP-binding proteins expressed in electromotor neurons.
The cDNAs encoding seven low molecular weight (LMW) GTP-binding proteins were isolated from an electrode lobe library of the marine ray Discopyge ommata. Four were assigned as the ray homologues of previously identified LMW GTP-binding proteins rab1, ral, Krev, and rho. Three others showed unique sequences, including two exhibiting significant similarity to the yeast SEC4 protein. Northern analysis indicated that several of the transcripts are enriched in neural tissues with a moderate level of expression in cardiac muscle. This tissue distribution was corroborated with affinity purified antibodies against the LMW GTP-binding proteins. Subcellular fractionation revealed that the proteins co-purify with cholinergic synaptic vesicles. Immunohistochemical analysis confirms this localization. At least two of the proteins, oral and o-rho, are localized to the pre-synaptic terminals.
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A synaptic vesicle specific GTP-binding protein from ray electric organ.
A cDNA encoding a synaptic vesicle associated GTP-binding protein was identified by screening a lambda gt11 expression library derived from the electric lobe of Discopyge ommata with polyclonal antibodies recognizing vesicle-specific proteins of Mr 25,000. Nucleotide sequence analysis defines an open reading frame of 218 amino acids. The protein belongs to the ras superfamily and shares about 75% amino acid identity with smg-25A, B and C identified in bovine brain and rab3A characterized in rat brain. Northern blot analysis revealed a 4.5 kb transcript present only in neural tissues, the highest level of expression being observed in electric lobe. Western blot analysis of total tissue homogenates derived from D. ommata detected the protein in electric organ, forebrain and to a lesser extent in electric lobe and spinal cord. No immunoreactivity was detected in non-neuronal tissues. Blotting of subcellular fractions derived from electric ray electric organ revealed that the GTP-binding protein co-purifies with synaptic vesicles. The neural specific expression and the localization to synaptic vesicles suggest a role of this protein in synaptic vesicle trafficking and targeting.
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The rat brain synucleins; family of proteins transiently associated with neuronal membrane.
The synuclein cDNA was previously cloned from Torpedo califonica using an antiserum against purified synaptic vesicles. Here we show that this gene hybridizes to several clones in a rat brain cDNA library. These clones represent cDNAs coding for different proteins, thus determining a family. These proteins display similar organization to the Torpedo protein. The homology resides within the repetition of 7 amino acids. The diversity is generated by alternative splicing as suggested by Southern analysis, as well as by sequence analysis. These proteins are specifically expressed in the rat brain. In situ hybridization shows expression of synuclein mRNAs in discrete areas of the brain, the hippocampus is the most intensely stained area. This localization is reminiscent of those molecules involved in phosphoinositols second messenger pathways. Synuclein proteins are present in rat brain in dual form--a small form which is soluble and a large form which is associated with synaptosomal membranes in a regulated fashion.
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Structure and expression of a rat agrin.
Agrin is a component of the basal lamina that causes the aggregation of acetylcholine receptors on cultured muscle fibers. An agrin cDNA clone isolated from electromotor neurons of a marine ray was used to characterize the corresponding cDNAs from a rat embryonic spinal cord library. Analysis of a set of clones predicts a 1940 amino acid protein containing 141 cysteine residues. The predicted protein has nine domains homologous to protease inhibitors, a region similar to domain III of laminin, and four epidermal growth factor repeats. The agrin gene is expressed in rat embryonic nervous system and muscle. The rat agrin protein is concentrated at synapses, where it may play a role in development and regeneration.
Agrin↗
Differential expression of the p65 gene family.
The genome of the marine ray Discopyge ommata contains at least three p65-related genes. o-p65-A is 84% identical, o-p65-B is 78% identical, and o-p65-C is only 41% identical to a previously characterized rat p65. The cytoplasmic domain, particularly the two regions that are similar to the regulatory domain of protein kinase C, are most highly conserved. The three genes are expressed in different but overlapping patterns in the central nervous system. o-p65-A immunoreactivity is found predominantly in forebrain, cerebellum, and neuroendocrine cells, while o-p65-B immunoreactivity is predominantly localized to the spinal cord, brainstem, and midbrain. Many synaptic vesicle proteins are members of small gene families that are differentially expressed, resulting in several unique combinations of these molecules in specific brain regions.
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Biosynthesis and sorting of neuropeptides.
As part of the secretory process neuropeptides are sorted from other cellular compartments and concentrated in vesicles. The vesicles are transported to release sites and stored, awaiting the proper signal for exocytosis. Regulation of the packaging process has many implications for neuropeptide function.
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