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GTP-Binding ProteinsGuanosine Triphosphaterab GTP-Binding ProteinsGTP Phosphohydrolasesrab5 GTP-Binding Proteinsrab3 GTP-Binding ProteinsGuanosine DiphosphateMolecular Sequence DataMonomeric GTP-Binding ProteinsAmino Acid SequenceGuanosine 5'-O-(3-Thiotriphosphate)rab4 GTP-Binding ProteinsADP-Ribosylation Factorsrab1 GTP-Binding ProteinsPertussis ToxinProtein BindingVirulence Factors, Bordetellarho GTP-Binding ProteinsADP Ribose TransferasesAdenosine Diphosphate RiboseBotulinum ToxinsBase SequenceThionucleotidesSequence Homology, Amino AcidGTPase-Activating ProteinsGuanine Nucleotide Exchange Factorsrab3A GTP-Binding ProteinCloning, Molecularras ProteinsMutationrhoA GTP-Binding Proteinrab2 GTP-Binding ProteinKineticsSignal TransductionPoly(A)-Binding ProteinsCell MembraneCarrier ProteinsSaccharomyces cerevisiaeCattleBinding SitesGTP CyclohydrolaseTacrolimus Binding ProteinsGuanine NucleotidesProtein Structure, TertiaryHydrolysisPoly(A)-Binding Protein IDNA-Binding ProteinsCell LineRecombinant ProteinsGuanylyl ImidodiphosphateInsulin-Like Growth Factor Binding ProteinsRecombinant Fusion ProteinsRNA-Binding ProteinsEscherichia coliModels, MolecularEndosomesRNA, MessengerProtein TransportMembrane ProteinsFatty Acid-Binding ProteinsTwo-Hybrid System TechniquesNuclear ProteinsHeLa CellsGuanosine MonophosphateTransfectionADP-Ribosylation Factor 1Peptide Elongation Factor TuSequence AlignmentBacterial ProteinsTransducinMutagenesis, Site-DirectedProtein PrenylationTransglutaminasesDynaminsPoly(A)-Binding Protein IIGuanine Nucleotide Dissociation InhibitorsPeptide Elongation Factor GGTP Phosphohydrolase-Linked Elongation FactorsDNA, ComplementaryEndocytosisProto-Oncogene Proteins p21(ras)Cells, CulturedProtein ConformationElectrophoresis, Polyacrylamide GelMolecular WeightCalcium-Binding ProteinsInsulin-Like Growth Factor Binding Protein 3Periplasmic Binding ProteinsGene Expression RegulationTranscription FactorsBlotting, WesternAdenylosuccinate SynthaseDNA PrimersPhosphorylationBiological TransportCalciumPeptide Elongation FactorsTubulinProteinsDNA

Aczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin. (1/87)

Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys(4) zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.  (+info)

Piccolo, a presynaptic zinc finger protein structurally related to bassoon. (2/87)

Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.  (+info)

Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. (3/87)

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.  (+info)

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. (4/87)

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.  (+info)

Binding of Rab3A to synaptic vesicles. (5/87)

Prenylated Rab GTPases cycle between membrane-bound and soluble forms. Membrane-bound GDP-Rabs interact with GDP dissociation inhibitor (GDI), resulting in the dissociation of a Rab.GDI complex, which in turn serves as a precursor for the membrane re-association of Rabs. We have now characterized the binding of Rab3A to synaptic vesicles in vitro using either purified complexes or rat brain cytosol as source for GDI.Rab3A. Binding of Rab3A results in the immediate release of GDI from the membrane. Furthermore, binding does not require the presence of additional guanine nucleotides (GDP or GTP) or of cytosolic factors. Although nucleotide exchange follows binding, binding is initially reversible, suggesting that binding of GDP-Rab3A and nucleotide exchange are separate and independent events. Comparison with the binding of Rab1B revealed that both Rab proteins bind preferentially to their respective resident membranes although some promiscuity was observable. Binding is saturable and involves a protease-sensitive binding site that is tightly associated with the vesicle membrane.  (+info)

Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP. (6/87)

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.  (+info)

Cyclosporine A-induced hypertension involves synapsin in renal sensory nerve endings. (7/87)

The calcineurin inhibitor cyclosporine A (CsA) has emerged as a major cause of secondary hypertension in humans, but the underlying pathogenetic mechanisms have remained enigmatic. Synapsins are a family of synaptic vesicle phosphoproteins that are essential for normal regulation of neurotransmitter release at synapses. In addition to synaptic vesicles, synapsins and other vesicle proteins are found on microvesicles in sensory nerve endings in peripheral tissues. However, the functions of the sensory microvesicles in general, and of synapsins in particular, are unknown. We now demonstrate in a mouse model that CsA raises blood pressure by stimulating renal sensory nerve endings that contain synapsin-positive microvesicles. In knockout mice lacking synapsin I and II, sensory nerve endings are normally developed but not stimulated by CsA whereas a control stimulus, capsaicin, is fully active. The reflex activation of efferent sympathetic nerve activity and the increase in blood pressure by CsA seen in control are greatly attenuated in synapsin-deficient mice. These results provide a mechanistic explanation for CsA-induced acute hypertension and suggest that synapsins could serve as a drug target in this refractory condition. Furthermore, these data establish evidence that synapsin-containing sensory microvesicles perform an essential role in sensory transduction and suggest a role for synapsin phosphorylation in this process.  (+info)

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor. (8/87)

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.  (+info)