G2Cdb::Gene report

Gene id
G00001468
Gene symbol
RAB3A (HGNC)
Species
Homo sapiens
Description
RAB3A, member RAS oncogene family
Orthologue
G00000219 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000071654 (Vega human gene)
Gene
ENSG00000105649 (Ensembl human gene)
5864 (Entrez Gene)
21 (G2Cdb plasticity & disease)
RAB3A (GeneCards)
Literature
179490 (OMIM)
Marker Symbol
HGNC:9777 (HGNC)
Protein Sequence
P20336 (UniProt)

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000159: Mental retardation Y Y (16584842) Single nucleotide polymorphism (SNP) N
D00000177: Autism Y Y (15005721) Unknown (?) N

References

  • Variants in the RAB3A gene are not associated with mental retardation in the Chinese population.

    Sun Y, Zhang F, Gao J, Gao X, Guo T, Shi Y, Tang W, Li S, Zheng Z, Zheng Y, Li X, Feng G and He L

    Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China.

    Mental retardation is a common form of cognitive impairment among children. The underlying causes of mental retardation are extremely heterogeneous, and include significant genetic factors. The coexistence of neuropathology and cognitive deficits supports the view that mental retardation is a disorder of brain development and plasticity. Rab3A, a member of the Rab small G protein family, is a key molecule in modulating basal neurotransmission and contributes to synaptic plasticity. The RAB3A gene is located on chromosome 19p13.11, near a region shown by a linkage study to be involved in the etiology of mental retardation. Because of both its function and chromosomal location, RAB3A is a potential candidate susceptibility gene for mental retardation. To investigate the possible genetic contribution of the RAB3A gene, we performed a case-control association study focused on the Han population of northwestern China using four common SNPs in the gene (rs7259012, rs17683539, rs2271882, and rs874628). Pairwise linkage disequilibrium analysis showed that the four SNPs were in linkage disequilibrium. However, there were no significant differences of either allele or genotype frequencies at any of the SNPs nor any significant differences in haplotype distributions between cases and controls. In conclusion, we have found no evidence for RAB3A conferring susceptibility on mental retardation in the Han Chinese population.

    Neuroscience letters 2006;401;1-2;114-8

  • DNA variants in the human RAB3A gene are not associated with autism.

    D'Adamo P, Bacchelli E, Blasi F, Lipp HP, Toniolo D and Maestrini E

    Institute of Anatomy, University of Zurich, Switzerland. p.dadamo@anatom.unizh.ch.

    Mutation screening of the RAB3A gene in 47 individuals with autism provided no evidence that DNA variants in this gene are associated with autism. Since Rab3a constitutive knockout mice react to novel stimuli with hyperactivity, a further search for association of RAB3A DNA variants with other neurobehavioral disorders such as attention deficit/hyperactivity disorder appears justified.

    Funded by: Telethon: E.1007, GGP030192

    Genes, brain, and behavior 2004;3;2;123-4

Literature (49)

Pubmed - other

  • APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle.

    Szodorai A, Kuan YH, Hunzelmann S, Engel U, Sakane A, Sasaki T, Takai Y, Kirsch J, Müller U, Beyreuther K, Brady S, Morfini G and Kins S

    Technical University of Kaiserslautern, Department of Human Biology and Human Genetics, D-67663 Kaiserslautern, Germany.

    The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by beta- and gamma-secretases leading to accumulation of beta-amyloid (Abeta) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by alpha-secretases prevents Abeta generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by alpha-secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.

    Funded by: NINDS NIH HHS: NS23320, NS23868, NS41170, NS43408, R01 NS023320, R01 NS023868, R01 NS023868-22, R01 NS023868-22S2, R01 NS041170, R01 NS043408, R56 NS023868

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;46;14534-44

  • Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis.

    Branham MT, Bustos MA, De Blas GA, Rehmann H, Zarelli VE, Treviño CL, Darszon A, Mayorga LS and Tomes CN

    Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología-Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina.

    Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2'-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.

    The Journal of biological chemistry 2009;284;37;24825-39

  • Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes.

    Figueiredo AC, Wasmeier C, Tarafder AK, Ramalho JS, Baron RA and Seabra MC

    Molecular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom.

    Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

    Funded by: Wellcome Trust: 075498

    The Journal of biological chemistry 2008;283;34;23209-16

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm.

    Lopez CI, Belmonte SA, De Blas GA and Mayorga LS

    Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.

    The acrosome reaction is a regulated Ca2+-dependent secretion event required for sperm-egg interaction. Previous studies indicate that the process requires Rab3-dependent tethering of membranes, SNARE complex assembly, and Ca2+-mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane-permeant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine-containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine-rich peptide, were able to translocate into sperm. Moreover, membrane-permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplasmic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocytosis. In conclusion, arginine-containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;14;4121-30

  • Variants in the RAB3A gene are not associated with mental retardation in the Chinese population.

    Sun Y, Zhang F, Gao J, Gao X, Guo T, Shi Y, Tang W, Li S, Zheng Z, Zheng Y, Li X, Feng G and He L

    Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China.

    Mental retardation is a common form of cognitive impairment among children. The underlying causes of mental retardation are extremely heterogeneous, and include significant genetic factors. The coexistence of neuropathology and cognitive deficits supports the view that mental retardation is a disorder of brain development and plasticity. Rab3A, a member of the Rab small G protein family, is a key molecule in modulating basal neurotransmission and contributes to synaptic plasticity. The RAB3A gene is located on chromosome 19p13.11, near a region shown by a linkage study to be involved in the etiology of mental retardation. Because of both its function and chromosomal location, RAB3A is a potential candidate susceptibility gene for mental retardation. To investigate the possible genetic contribution of the RAB3A gene, we performed a case-control association study focused on the Han population of northwestern China using four common SNPs in the gene (rs7259012, rs17683539, rs2271882, and rs874628). Pairwise linkage disequilibrium analysis showed that the four SNPs were in linkage disequilibrium. However, there were no significant differences of either allele or genotype frequencies at any of the SNPs nor any significant differences in haplotype distributions between cases and controls. In conclusion, we have found no evidence for RAB3A conferring susceptibility on mental retardation in the Han Chinese population.

    Neuroscience letters 2006;401;1-2;114-8

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

    Belmonte SA, López CI, Roggero CM, De Blas GA, Tomes CN and Mayorga LS

    Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, 5500 Mendoza, Argentina.

    The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

    Developmental biology 2005;285;2;393-408

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells.

    Matsumoto M, Miki T, Shibasaki T, Kawaguchi M, Shinozaki H, Nio J, Saraya A, Koseki H, Miyazaki M, Iwanaga T and Seino S

    Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

    Rab3 is a subfamily of the small GTP-binding protein Rab family and plays an important role in exocytosis. Several potential effectors of Rab3, including rabphilin3 and Rims (Rim1 and Rim2), have been isolated and characterized. Noc2 was identified originally in endocrine pancreas as a molecule homologous to rabphilin3, but its role in exocytosis is unclear. To clarify the physiological function of Noc2 directly, we have generated Noc2 knockout (Noc2(-/-)) mice. Glucose intolerance with impaired insulin secretion was induced in vivo by acute stress in Noc2(-/-) mice, but not in wild-type (Noc2(+/+)) mice. Ca(2+)-triggered insulin secretion from pancreatic isles of Noc2(-/-) mice was markedly impaired, but was completely restored by treatment with pertussis toxin, which inhibits inhibitory G protein Gi/o signaling. In addition, the inhibitory effect of clonidine, an alpha(2)-adrenoreceptor agonist, on insulin secretion was significantly greater in Noc2(-/-) islets than in Noc2(+/+) islets. Impaired Ca(2+)-triggered insulin secretion was rescued by adenovirus gene transfer of wild-type Noc2 but not by that of mutant Noc2, which does not bind to Rab3. Accordingly, Noc2 positively regulates insulin secretion from endocrine pancreas by inhibiting Gi/o signaling, and the interaction of Noc2 and Rab3 is required for the effect. Interestingly, we also found a marked accumulation of secretory granules in various exocrine cells of Noc2(-/-) mice, especially in exocrine pancreas with no amylase response to stimuli. Thus, Noc2, a critical effector of Rab3, is essential in normal regulation of exocytosis in both endocrine and exocrine cells.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;22;8313-8

  • The DNA sequence and biology of human chromosome 19.

    Grimwood J, Gordon LA, Olsen A, Terry A, Schmutz J, Lamerdin J, Hellsten U, Goodstein D, Couronne O, Tran-Gyamfi M, Aerts A, Altherr M, Ashworth L, Bajorek E, Black S, Branscomb E, Caenepeel S, Carrano A, Caoile C, Chan YM, Christensen M, Cleland CA, Copeland A, Dalin E, Dehal P, Denys M, Detter JC, Escobar J, Flowers D, Fotopulos D, Garcia C, Georgescu AM, Glavina T, Gomez M, Gonzales E, Groza M, Hammon N, Hawkins T, Haydu L, Ho I, Huang W, Israni S, Jett J, Kadner K, Kimball H, Kobayashi A, Larionov V, Leem SH, Lopez F, Lou Y, Lowry S, Malfatti S, Martinez D, McCready P, Medina C, Morgan J, Nelson K, Nolan M, Ovcharenko I, Pitluck S, Pollard M, Popkie AP, Predki P, Quan G, Ramirez L, Rash S, Retterer J, Rodriguez A, Rogers S, Salamov A, Salazar A, She X, Smith D, Slezak T, Solovyev V, Thayer N, Tice H, Tsai M, Ustaszewska A, Vo N, Wagner M, Wheeler J, Wu K, Xie G, Yang J, Dubchak I, Furey TS, DeJong P, Dickson M, Gordon D, Eichler EE, Pennacchio LA, Richardson P, Stubbs L, Rokhsar DS, Myers RM, Rubin EM and Lucas SM

    Stanford Human Genome Center, Department of Genetics, Stanford University School of Medicine, 975 California Avenue, Palo Alto, California 94304, USA. jane@shgc.stanford.edu

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

    Nature 2004;428;6982;529-35

  • DNA variants in the human RAB3A gene are not associated with autism.

    D'Adamo P, Bacchelli E, Blasi F, Lipp HP, Toniolo D and Maestrini E

    Institute of Anatomy, University of Zurich, Switzerland. p.dadamo@anatom.unizh.ch.

    Mutation screening of the RAB3A gene in 47 individuals with autism provided no evidence that DNA variants in this gene are associated with autism. Since Rab3a constitutive knockout mice react to novel stimuli with hyperactivity, a further search for association of RAB3A DNA variants with other neurobehavioral disorders such as attention deficit/hyperactivity disorder appears justified.

    Funded by: Telethon: E.1007, GGP030192

    Genes, brain, and behavior 2004;3;2;123-4

  • Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release.

    Takao-Rikitsu E, Mochida S, Inoue E, Deguchi-Tawarada M, Inoue M, Ohtsuka T and Takai Y

    KAN Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8815, Japan.

    We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.

    The Journal of cell biology 2004;164;2;301-11

  • Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.

    Rastaldi MP, Armelloni S, Berra S, Li M, Pesaresi M, Poczewski H, Langer B, Kerjaschki D, Henger A, Blattner SM, Kretzler M, Wanke R and D'Amico G

    Renal Immunopathology Laboratory, Associazione Nuova Nefrologia, c/o San Carlo Borromeo Hospital, Via Pio II, 20153 Milan, Italy. mp.rastaldi@oscb.sined.net

    Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormone-transgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes, further supports similarities between cytoskeletal and vesicular organization of podocytes and neurons. The altered expression observed in mouse and human proteinuric diseases suggests a possible role for these molecules in glomerulopathies.

    The American journal of pathology 2003;163;3;889-99

  • Distinct Rab binding specificity of Rim1, Rim2, rabphilin, and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2.

    Fukuda M

    Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp

    Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.

    The Journal of biological chemistry 2003;278;17;15373-80

  • Slp4-a/granuphilin-a inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells.

    Fukuda M

    Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp

    Slp4-a (synaptotagmin-like protein 4-a)/granuphilin-a is specifically localized on dense-core vesicles in PC12 cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A via the N-terminal Slp homology domain (SHD) (Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y., and Kuroda, T. S. (2002) J. Biol. Chem. 277, 39673-39678). However, the mechanism of the inhibition by Slp4-a has never been elucidated at the molecular level and is still a matter of controversy. In this study, I discovered an unexpected biochemical property of Slp4-a, that Slp4-a, but not other Rab27 effectors reported thus far, is capable of interacting with both Rab27A(T23N), a dominant negative form that mimics the GDP-bound form, and Rab27A(Q78L), a dominant active form that mimics the GTP-bound form, whereas Slp4-a specifically recognizes the GTP-bound form of Rab3A and Rab8A and does not recognize their GDP-bound form. I show by deletion and mutation analyses that the TGDWFY sequence in SHD2 is essential for Rab27A(T23N) binding, whereas SHD1 is involved in Rab27A(Q78L) binding. I further show by immunoprecipitation and cotransfection assays that Munc18-1, but not syntaxin IA, directly interacts with the C-terminal domain of Slp4-a in a Rab27A-independent manner. Expression of Slp4-a mutants that lack Rab27A(T23N) binding activity (i.e. specific binding to Rab27A(Q78L)) completely reverses the inhibitory effect of the wild-type Slp4-a on high KCl-dependent neuropeptide Y secretion in PC12 cells. The results strongly indicate that interaction of Slp4-a with the GDP-bound form of Rab27A, not with syntaxin IA or Munc18-1, is the primary reason that Slp4-a expression inhibits dense core vesicle exocytosis in PC12 cells.

    The Journal of biological chemistry 2003;278;17;15390-6

  • Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface.

    Pereira-Leal JB, Strom M, Godfrey RF and Seabra MC

    Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, UK.

    Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.

    Biochemical and biophysical research communications 2003;301;1;92-7

  • Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells.

    Fukuda M, Kanno E, Saegusa C, Ogata Y and Kuroda TS

    Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp

    Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic beta-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542-28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitro and in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218). How Slp4-a.Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a.Rab8 and Slp4-a.Rab27A complexes, but not Slp4-a.Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.

    The Journal of biological chemistry 2002;277;42;39673-8

  • Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse.

    Kapfhamer D, Valladares O, Sun Y, Nolan PM, Rux JJ, Arnold SE, Veasey SC and Bućan M

    Center for Neurobiology of Behavior of the Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.

    Nature genetics 2002;32;2;290-5

  • Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains.

    Kuroda TS, Fukuda M, Ariga H and Mikoshiba K

    Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

    Synaptotagmin-like proteins 1-4 (Slp1-4) are new members of the carboxyl-terminal-type (C-type) tandem C2 proteins and are classified as a subfamily distinct from the synaptotagmin and the Doc2 families, because the Slp family contains a unique homology domain at the amino terminus, referred to as the Slp homology domain (SHD). We previously showed that the SHD functions as a binding site for Rab27A, which is associated with human hemophagocytic syndrome (Griscelli syndrome) [J. Biol. Chem. 277 (2002) 9212; J. Biol. Chem. 277 (2002) 12432]. In the present study, we identified a novel member of the Slp family, Slp5. The same as other Slp family members, the SHD of Slp5 preferentially interacted with the GTP-bound form of Rab27A and marginally with Rab3A and Rab6A, both in vitro and in intact cells, but not with other Rabs tested (Rab1, Rab2, Rab4A, Rab5A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). However, unlike other members of the Slp family, expression of Slp5 mRNA was highly restricted to human placenta and liver. Expression of Slp5 protein and in vivo association of Slp5 with Rab27A in the mouse liver were further confirmed by immunoprecipitation. The results suggest that Slp5 might be involved in Rab27A-dependent membrane trafficking in specific tissues.

    Biochemical and biophysical research communications 2002;293;3;899-906

  • Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family.

    Nagano F, Kawabe H, Nakanishi H, Shinohara M, Deguchi-Tawarada M, Takeuchi M, Sasaki T and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.

    Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

    The Journal of biological chemistry 2002;277;12;9629-32

  • The Slp homology domain of synaptotagmin-like proteins 1-4 and Slac2 functions as a novel Rab27A binding domain.

    Kuroda TS, Fukuda M, Ariga H and Mikoshiba K

    Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama, Japan.

    rab27A, which encodes a small GTP-binding protein, was recently identified as a gene in which mutations caused human hemophagocytic syndrome (Griscelli syndrome) and ashen mice, which exhibit defects in melanosome transport as well as in regulated granule exocytosis in cytotoxic T lymphocytes. However, little is known about the molecular mechanism of Rab27A-dependent membrane trafficking or the specific effector molecules of Rab27A. In this study, we discovered that the Slp (synaptotagmin-like protein) homology domain (SHD) of Slp1--3 and Slac2-a/b specifically and directly binds the GTP-bound form of Rab27A both in vitro and in intact cells but not of the other Rabs tested (Rab1, Rab2, Rab3A, Rab4, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). Immunocytochemical analysis revealed that Slp2 (or Slp1) colocalized with Rab27A in the melanosomes of melanoma cells. Slp2 and Rab27A were distributed to the periphery of the cells (especially at the dendritic tips) in the wild-type melanoma cells, whereas they accumulated in the perinuclear region in the melanosome transport-defective cells (S91/Cloudman). These results strongly indicated that the SHD of Slp1--3 and Slac2 functions as an in vivo Rab27A binding domain.

    The Journal of biological chemistry 2002;277;11;9212-8

  • SNARE complex assembly is required for human sperm acrosome reaction.

    Tomes CN, Michaut M, De Blas G, Visconti P, Matti U and Mayorga LS

    Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. ctomes@fmed2.uncu.edu.ar

    Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca(2+) is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+). Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.

    Funded by: NICHD NIH HHS: HD 38082

    Developmental biology 2002;243;2;326-38

  • The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules.

    Yi Z, Yokota H, Torii S, Aoki T, Hosaka M, Zhao S, Takata K, Takeuchi T and Izumi T

    Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.

    Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

    Molecular and cellular biology 2002;22;6;1858-67

  • Subcellular distribution and function of Rab3A-D in pancreatic acinar AR42J cells.

    Piiper A, Leser J, Lutz MP, Beil M and Zeuzem S

    Department of Medicine II, University of Frankfurt/Main, Frankfurt, 60590, Germany. Piiper@em.uni-frankfurt.de

    Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J cells.

    Biochemical and biophysical research communications 2001;287;3;746-51

  • GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase.

    Luo HR, Saiardi A, Nagata E, Ye K, Yu H, Jung TS, Luo X, Jain S, Sawa A and Snyder SH

    Department of Neuroscience, School of Medicine, Johns Hopkins University, 725 N. Wolfe Street, Baltimore, MD 21205, USA.

    Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.

    Funded by: NIDA NIH HHS: DA-00075; NIMH NIH HHS: MH-18501

    Neuron 2001;31;3;439-51

  • The scaffolding protein CASK mediates the interaction between rabphilin3a and beta-neurexins.

    Zhang Y, Luan Z, Liu A and Hu G

    Max-Planck Guest Laboratory, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320, Yue-Yang Road, 200031, Shanghai, PR China.

    CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl-terminus of beta-neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase-like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein-protein interaction. By a yeast two-hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull-down and co-immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by beta-neurexins via the interaction between rabphilin3a-CASK-beta-neurexins.

    FEBS letters 2001;497;2-3;99-102

  • Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.

    Betz A, Thakur P, Junge HJ, Ashery U, Rhee JS, Scheuss V, Rosenmund C, Rettig J and Brose N

    Max-Planck-Institut, für experimentelle Medizin, Abteilung Neurogenetik, AG Molekulare Neurobiologie, Hermann-Rein-Str. 3, D-37075, Göttingen, Germany.

    Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

    Neuron 2001;30;1;183-96

  • A direct inhibitory role for the Rab3-specific effector, Noc2, in Ca2+-regulated exocytosis in neuroendocrine cells.

    Haynes LP, Evans GJ, Morgan A and Burgoyne RD

    Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.

    Rab proteins comprise a family of GTPases, conserved from yeast to mammals, which are integral components of membrane trafficking pathways. Rab3A is a neural/neuroendocrine-specific member of the Rab family involved in Ca(2+) -regulated exocytosis, where it functions in an inhibitory capacity controlling recruitment of secretory vesicles into a releasable pool at the plasma membrane. The effector by which Rab3A exerts its inhibitory effect is unclear as the Rab3A effectors Rabphilin and RIM have been excluded from for this role. One putative Rab3A effector in dense-core granule exocytosis is the cytosolic zinc finger protein, Noc2. We have established that overexpression of Noc2 in PC12 cells has a direct inhibitory effect upon Ca(2+)-triggered exocytosis in permeabilized cells. We demonstrate specific nucleotide-dependent binding of Noc2 to Rab3A and show that the inhibition of exocytosis is dependent upon this interaction since Rab3A binding-deficient mutants of Noc2 do not inhibit exocytosis. We propose that Noc2 may be a negative effector for Rab3A in regulated exocytosis of dense-core granules from endocrine cells.

    The Journal of biological chemistry 2001;276;13;9726-32

  • Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

    Tabellini G, Baldini G, Baldini G, Bortul R, Bareggi R, Narducci P and Martelli AM

    Dipartimento di Morfologia Umana Normale, Università degli Studi di Trieste, Italy.

    We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.

    Funded by: NIDDK NIH HHS: R01-DK53293

    European journal of histochemistry : EJH 2001;45;4;347-56

  • Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP.

    Clabecq A, Henry JP and Darchen F

    CNRS UPR 1929, Institut de Biologie Physico-Chimique, 13 rue P. et M. Curie, 75005 Paris, France.

    Small G proteins of the Rab family are regulators of intracellular vesicle traffic. Their intrinsic rate of GTP hydrolysis is very low but is enhanced by specific GTPase-activating proteins (GAPs) that switch G proteins to their inactive form. We have characterized the activity of recombinant Rab3-GAP on Rab3A in solution. The K(m) and K(d) values (75 microm) indicate a low affinity of Rab3-GAP for its substrate. The affinity is higher for the transition state analog Rab3A:GDP:AlF(x) (15 microm). The k(cat) (1 s(-)(1)) is within the range of values reported for other GAPs. A mutation in the switch I region of Rab3A disrupted the interaction with Rab3-GAP. Furthermore, Rabphilin, a putative target of Rab3, inhibited the activity of Rab3-GAP on Rab3. Therefore, the Rab3-GAP-binding site involves the switch I region of Rab3 and overlaps with the Rabphilin-binding domain. Substitution of a single arginine residue (Arg-728) of Rab3-GAP disrupted its catalytic activity but not its interaction with Rab3A. We propose that Rab3-GAP, like Ras- and Rho-GAPs, stabilizes the transition state of Rab3 and provides a critical arginine residue to accelerate the GTPase reaction.

    The Journal of biological chemistry 2000;275;41;31786-91

  • Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19p13.1, between RAB3A and JUND.

    Sullivan M, Olsen AS and Houslay MD

    Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, UK.

    PDE4C is one of four mammalian genes that encode multiple PDE4 cyclic AMP-specific phosphodiesterase isoforms that are inhibited by rolipram. Fluorescent in situ hybridisation localised PDE4C to the p13.1 region of human chromosome 19. Overlapping cosmid clones spanning the human PDE4C gene were identified and characterised. Analysis of this locus indicated that the PDE4C gene spans at least 38 kb, consists of at least 18 exons, and contains the marker D19S212 within an intron. Comparison of published human PDE4C cDNA sequences with those of the genomic DNA identified four alternatively spliced exons and the possibility that the PDE4C locus contains at least three alternative promoters. PDE4C-containing cosmids also contained the genes for the growth regulatory transcription factor, JUND, and the mini guanine nucleotide regulatory protein, RAB3A. The RAB3A gene was shown to consist of 5 exons spanning 7.9 kb, while the JUND gene was found to contain no introns. Analysis of cosmids containing PDE4C, JUND, and RAB3A showed that 27 kb separate JUND and PDE4C, while only 3.7 kb separate PDE4C and RAB3A. The three genes share the same orientation of transcription and are arranged in the order cen- 5'- JUND-PDE4C-RAB3A-3'-tel.

    Cellular signalling 1999;11;10;735-42

  • Phosphatidic acid is important to the translocation of Rab3A from the cytosol to phospholipid membranes.

    Jung YJ, Lee TH, Lee JY, Kim JH and Park JB

    Department of Biochemistry, College of Medicine, Hallym University, Chun-Chon, Kang-Won Do, South Korea.

    RAB3A, a Ras-related GTP-binding protein, is found in a rat neuronal cytosol in the form of complex with RabGDI or located on the synaptic vesicles and it cycles between the cytosol and the membranes. However, the regulatory mechanism of the translocation of Rab3A has not been clearly understood. To understand the mechanism of the translocation of Rab3A from the cytosol to the membranes, we examined which membranenous phospholipid is required for the translocation of Rab3A. Phosphatidic acid (PA) was found to be required for the translocation of Rab3A to the membranes and GTPgammaS stimulated the translocation of Rab3A.

    Neuroreport 1999;10;13;2859-63

  • Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A.

    Ostermeier C and Brunger AT

    The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.

    The small G protein Rab3A plays an important role in the regulation of neurotransmitter release. The crystal structure of activated Rab3A/GTP/Mg2+ bound to the effector domain of rabphilin-3A was solved to 2.6 A resolution. Rabphilin-3A contacts Rab3A in two distinct areas. The first interface involves the Rab3A switch I and switch II regions, which are sensitive to the nucleotide-binding state of Rab3A. The second interface consists of a deep pocket in Rab3A that interacts with a SGAWFF structural element of rabphilin-3A. Sequence and structure analysis, and biochemical data suggest that this pocket, or Rab complementarity-determining region (RabCDR), establishes a specific interaction between each Rab protein and its effectors. RabCDRs could be major determinants of effector specificity during vesicle trafficking and fusion.

    Cell 1999;96;3;363-74

  • Isolation and characterization of a dual prenylated Rab and VAMP2 receptor.

    Martincic I, Peralta ME and Ngsee JK

    Department of Medicine and Biochemistry, Loeb Research Institute, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada.

    Rab GTPases have been implicated in intracellular vesicle trafficking. Using the yeast two-hybrid screen, we have isolated a rat clone that interacts with Rab3A as well as with Rab1. The gene encodes a 20.6-kDa protein with two extensive hydrophobic domains and is broadly expressed in all tissues. This protein binds to prenylated Rab GTPases but not to other small Ras-like GTPases such as the Rho/Rac family. This prenylated Rab acceptor (PRA1) also binds specifically to the synaptic vesicle protein VAMP2 (or synaptobrevin II) but shows no affinity for VAMP1 or cellubrevin in both the yeast two-hybrid system and in vitro binding assays. This specificity resides, in part, in the proline-rich domain of VAMP2 as a chimera containing this domain of VAMP2 fused to VAMP1 is able to bind to PRA1. The transmembrane domain of VAMP2 is also essential as its deletion abolished binding to PRA1. Replacement of the deleted VAMP2 transmembrane domain by a CAAX prenylation signal can not restore binding to PRA1. This interaction is therefore distinct from that required for VAMP2 binding to either syntaxin or both syntaxin and SNAP-25. Deletion analysis on PRA1 indicates that the critical Rab- and VAMP2-interacting residues reside in two regions: the amino-terminal residues 30-54 and the extreme carboxyl-terminal domain. This dual Rab and VAMP2 binding characteristic suggests that PRA1 may serve to link these two protein families in the control of vesicle docking and fusion.

    The Journal of biological chemistry 1997;272;43;26991-8

  • The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion.

    Geppert M, Goda Y, Stevens CF and Südhof TC

    Max Planck Institute for Experimental Medicine, Gottingen, Germany.

    The Rab family of low-molecular-mass GTP-binding proteins are thought to guide membrane fusion between a transport vesicle and the target membrane, and to determine the specificity of docking. The docking and fusion of vesicles is, however, a complex multistep reaction, and the precise point at which Rab proteins act in these sequential processes is unknown. In brain, the Rab protein Rab3A is specific to synaptic vesicles, whose exocytosis can be monitored with submillisecond resolution by following synaptic transmission. We have now determined the precise point at which Rab3A acts in the sequence of synaptic vesicle docking and fusion by using electrophysiological analysis of neurotransmitter release in Rab3A-deficient mice. Unexpectedly, the size of the readily releasable pool of vesicles is normal, whereas Ca2+-triggered fusion is altered in the absence of Rab3A in that a more-than-usual number of exocytic events occur within a brief time after arrival of the nerve impulse.

    Nature 1997;387;6635;810-4

  • An evolutionarily conserved domain in a subfamily of Rabs is crucial for the interaction with the guanyl nucleotide exchange factor Mss4.

    Burton JL, Slepnev V and De Camilli PV

    Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Mss4 is a guanine nucleotide exchange factor that specifically binds to, and promotes GDP-GTP exchange on, a subset of the Rab GTPases (Burton, J. L., Burns, M. E., Gatti, E., Augustine, G. J., and De Camilli, P. (1994) EMBO J. 13, 5547-5558). In order to identify the domain(s) of the GTPase that is important for this interaction, protein chimeras were constructed between Rab3a, which binds Mss4, and Rab5a, which does not bind Mss4. We have identified the amino-terminal portion of Rab3a as the Mss4-binding region, with the effector domain being critically required for binding and the flanking regions further enhancing the interaction. Sequence comparisons have revealed that Mss4-binding Rabs share more homology with each other than with Rabs that do not bind Mss4. The region of highest homology between these Rabs, which defines them as members of the same evolutionary branch within the Rab subfamily, coincides with the domain shown here to be critical for Mss4 binding. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a region that is highly conserved between Mss4 and its yeast homologue Dss4, completely abolished its property to bind to, and promote GDP-GTP exchange on, Rab3a. Thus, the preservation of the Mss4/Dss4-GTPase interaction appears to have been a critical factor in the evolution of this subset of Rab proteins.

    Funded by: NCI NIH HHS: CA46128

    The Journal of biological chemistry 1997;272;6;3663-8

  • Characterization of the interaction of the monomeric GTP-binding protein Rab3a with geranylgeranyl transferase II.

    Johannes L, Perez F, Laran-Chich MP, Henry JP and Darchen F

    Service de Neurobiologie Physico-Chimique, Centre National de la Recherche Scientifique UPR 9071, Paris, France.

    The monomeric GTP-binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells. Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C-terminus. The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II. We describe the cDNA cloning of the beta-subunit of human geranylgeranyl transferase II by means of the yeast two-hybrid system. The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the beta-subunit deficiency in the yeast strain ANY119. Furthermore, by means of the two-hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II. We find that the N-terminus, the effector loop, the hypervariable region of the C-terminus, and the geranylgeranyl-acceptor cysteines have roles in this interaction. The GDP-bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II.

    European journal of biochemistry 1996;239;2;362-8

  • Distinct functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells.

    Weber E, Jilling T and Kirk KL

    Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 35294, USA.

    Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells.

    Funded by: NIDDK NIH HHS: DK50830

    The Journal of biological chemistry 1996;271;12;6963-71

  • Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

    Brondyk WH, McKiernan CJ, Fortner KA, Stabila P, Holz RW and Macara IG

    Department of Pathology, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405-0068.

    Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.

    Funded by: NCI NIH HHS: R01 CA38888; NEI NIH HHS: F32 EY6411; NIDDK NIH HHS: R01 DK27959

    Molecular and cellular biology 1995;15;3;1137-43

  • Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

    Farnsworth CC, Seabra MC, Ericsson LH, Gelb MH and Glomset JA

    Howard Hughes Medical Institute, University of Washington, Seattle 98195.

    Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.

    Funded by: NCI NIH HHS: CA52874; NCRR NIH HHS: RR-00166; NHLBI NIH HHS: HL20498

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;25;11963-7

  • Functional identification of a vesicular acetylcholine transporter and its expression from a "cholinergic" gene locus.

    Erickson JD, Varoqui H, Schäfer MK, Modi W, Diebler MF, Weihe E, Rand J, Eiden LE, Bonner TI and Usdin TB

    Section on Molecular Neuroscience, National Institute of Mental Health, Bethesda, Maryland 20892.

    The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA library encoding this protein. The distribution of VAChT mRNA coincides with that reported for choline acetyltransferase (ChAT), the enzyme required for acetylcholine biosynthesis, in the peripheral and central cholinergic nervous systems. A human VAChT cDNA was used to localize the VAChT gene to chromosome 10q11.2, which is also the location of the ChAT gene. The entire sequence of the human VAChT cDNA is contained uninterrupted within the first intron of the ChAT gene locus. Transcription of VAChT and ChAT mRNA from the same or contiguous promoters within a single regulatory locus provides a previously undescribed genetic mechanism for coordinate regulation of two proteins whose expression is required to establish a mammalian neuronal phenotype.

    Funded by: NIGMS NIH HHS: R01 GM038679

    The Journal of biological chemistry 1994;269;35;21929-32

  • REP-2, a Rab escort protein encoded by the choroideremia-like gene.

    Cremers FP, Armstrong SA, Seabra MC, Brown MS and Goldstein JL

    Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Texas 75235-9046.

    Rab escort proteins (REPs) bind to newly synthesized Rab proteins and remain bound during and after the attachment of a geranylgeranyl (GG) group by the catalytic component of the Rab GG transferase. Transfer of the GG group is absolutely dependent on the participation of a REP. REP-1, the first characterized REP, is produced by a gene on the X chromosome that is defective in patients with choroideremia, a form of retinal degeneration. Cremers et al. (Cremers, F.P.M., Molloy, C. M., van de Pol, D. J. R., van den Hurk, J. A. J. M., Bach, I., Geurts van Kessel, A. H. M., and Ropers, H.-H. (1992) Hum. Mol. Genet. 1, 71-75) isolated a related gene, designated choroideremia-like, which encodes a protein that closely resembles REP-1. In the current studies, we produced REP-1 and REP-2 by recombinant DNA methods and showed that both proteins were approximately equal in facilitating the attachment of GG groups to several Rab proteins, including Rab1A, Rab5A, and Rab6. However, REP-2 was only 25% as active as REP-1 in supporting GG attachment to Rab3A and Rab3D. The low activity toward Rab3A was increased to that of Rab1A when the COOH-terminal 12 amino acids of Rab3A were replaced with the corresponding residues of Rab1A. We suggest that REP-2 substitutes for the absent function of REP-1 in nonretinal cells of patients with choroideremia, thus preventing cellular dysfunction throughout the body. In the retina, REP-2 may be only partially effective, leading eventually to retinal degeneration and blindness.

    Funded by: NHLBI NIH HHS: HL20948; NIGMS NIH HHS: GM08014

    The Journal of biological chemistry 1994;269;3;2111-7

  • The Rab3A GTPase interacts with multiple factors through the same effector domain. Mutational analysis of cross-linking of Rab3A to a putative target protein.

    McKiernan CJ, Brondyk WH and Macara IG

    Department of Pathology, University of Vermont Medical College, Burlington 05405-0068.

    Rab3A/smg25A is a small Ras-like guanine nucleotide binding protein implicated in the control of regulated secretion from cells. Rab3A is approximately 30% cytosolic and 70% associated with the membranes of secretory vesicles. It cross-links specifically to a rat brain membrane protein of about 85 kilodaltons (p85). To identify epitopes on Rab3A that are important for its interaction with this putative target protein, we have determined the effects of point mutations on the cross-linking efficiency of Rab3A to p85. Rab3A, which was preincubated with a non-hydrolyzable analog of GTP, cross-linked more efficiently to p85 than did Rab3A-GDP. Rab3A mutants that had decreased nucleotide binding also exhibited poor cross-linking to p85. Mutations in the effector domain, a site important for the interaction of Rab3A with its guanine nucleotide releasing factor, guanine nucleotide dissociation inhibitor, and GTPase-activating protein, eliminated the ability of Rab3A to cross-link to p85. However, short peptides corresponding to the effector domain did not reduce cross-linking efficiency when present at a concentration of 50 microM.

    Funded by: NCI NIH HHS: CA56300; NEI NIH HHS: F32EY06411

    The Journal of biological chemistry 1993;268;32;24449-52

  • Fluorescence in situ hybridization mapping of human chromosome 19: cytogenetic band location of 540 cosmids and 70 genes or DNA markers.

    Trask B, Fertitta A, Christensen M, Youngblom J, Bergmann A, Copeland A, de Jong P, Mohrenweiser H, Olsen A, Carrano A et al.

    Human Genome Center, Lawrence Livermore National Laboratory, Livermore, California 94550.

    We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19 cosmids on other chromosomes is also reported. Colony hybridization identified 142 cosmids that contain sequences representing genes or DNA markers that map to chromosome 19. FISH mapping of these cosmids sublocalizes a total of 70 genes and DNA markers on chromosome 19, revises the previously published map assignments of 2 genes, and narrows the location of over 20 markers.

    Funded by: NHGRI NIH HHS: HG-00256

    Genomics 1993;15;1;133-45

  • Isoprenoid modification of rab proteins terminating in CC or CXC motifs.

    Khosravi-Far R, Lutz RJ, Cox AD, Conroy L, Bourne JR, Sinensky M, Balch WE, Buss JE and Der CJ

    La Jolla Cancer Research Foundation, CA 92037.

    Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.

    Funded by: NCI NIH HHS: CA42978, CA52072, CA55008

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;14;6264-8

  • Chromosome mapping of the human ras-related rab3A gene to 19p13.2.

    Rousseau-Merck MF, Zahraoui A, Bernheim A, Touchot N, Miglierina R, Tavitian A and Berger R

    INSERM Unité 301, Hôpital Saint-Louis, Paris, France.

    The rab genes belong to one of the three main branches of the ras super family. The encoded rab proteins share 38 to 75% amino acid identity with the yeast YPT1 and SEC4 proteins. We used the human rab3A cDNA to map the corresponding gene on human chromosomes by chromosome sorting and in situ hybridization. Both techniques allowed the assignment of the rab3A gene to chromosome 19 with a regional localization on 19p13.2 obtained by in situ hybridization.

    Genomics 1989;5;4;694-8

  • The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion.

    Zahraoui A, Touchot N, Chardin P and Tavitian A

    Institut National de la Santé et de la Recherche Médicale U. 248, Faculté de Médecine Lariboisiere Saint-Louis Paris, France.

    Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.

    The Journal of biological chemistry 1989;264;21;12394-401

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000032 G2C Homo sapiens Pocklington H1 Human orthologues of cluster 1 (mouse) from Pocklington et al (2006) 21
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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