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S M Bajjalieh

Publications and source records attributed to S M Bajjalieh.

15 recordsLinked to original sources
PubMed2001

SV2 modulates the size of the readily releasable pool of secretory vesicles.

The exocytosis of neurotransmitters is regulated by calcium and is plastic - features that suggest specialized regulation of the basic membrane trafficking process. Here we show that Synaptic Vesicle Protein 2 (SV2), a protein specific to neurons and endocrine cells, is required to maintain a pool of vesicles available for calcium-stimulated exocytosis. Direct measures of exocytosis in adrenal chromaffin cells showed that the calcium-induced exocytotic burst, which operationally defines the readily releasable pool of vesicles, was significantly reduced in mice lacking SV2A. Burst kinetics were normal in cells from SV2A knockout animals, however, indicating that SV2 functions before the final events of fusion. Analyses of SDS-resistant SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion) attachment protein receptor) complexes in brain tissue showed that loss of SV2A was associated with fewer SDS-resistant complexes. Our observations indicate that SV2 may modulate the formation of protein complexes required for fusion and therefore the progression of vesicles to a fusion-competent state.

Adrenal Medulla↗
PubMed2000

Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin.

Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2.

Animals↗
PubMed1999

Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A).

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein common to all synaptic and endocrine vesicles. Unlike many proteins involved in synaptic exocytosis, SV2 has no homolog in yeast, indicating that it performs a function unique to secretion in higher eukaryotes. Although the structure and protein interactions of SV2 suggest multiple possible functions, its role in synaptic events remains unknown. To explore the function of SV2 in an in vivo context, we generated mice that do not express the primary SV2 isoform, SV2A, by using targeted gene disruption. Animals homozygous for the SV2A gene disruption appear normal at birth. However, they fail to grow, experience severe seizures, and die within 3 weeks, suggesting multiple neural and endocrine deficits. Electrophysiological studies of spontaneous inhibitory neurotransmission in the CA3 region of the hippocampus revealed that loss of SV2A leads to a reduction in action potential-dependent gamma-aminobutyric acid (GABA)ergic neurotransmission. In contrast, action potential-independent neurotransmission was normal. Analyses of synapse ultrastructure suggest that altered neurotransmission is not caused by changes in synapse density or morphology. These findings demonstrate that SV2A is an essential protein and implicate it in the control of exocytosis.

Animals↗
PubMed1999

Synaptic vesicle docking and fusion.

Neurotransmitter secretion shares many features with constitutive membrane trafficking. In both cases, vesicles are targeted to a specific acceptor membrane and fuse via a series of protein-protein interactions. Recent work has added to the list of protein complexes involved and is beginning to define the order in which they act. The rapid fusion, precise regulation and plasticity characteristic of synaptic exocytosis probably results from the addition of specialized regulators.

Animals↗
PubMed1996

Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin.

The identification and functional characterization of proteins localized to synaptic vesicles has contributed significantly to our understanding of neurotransmission. Studies of synaptic vesicle protein interactions have both led to the identification of novel synaptic proteins and suggested hypotheses of protein function. Synaptic vesicle protein 2 (SV2), is an integral membrane glycoprotein present in all synaptic vesicles. There are two characterized isoforms, SV2A and SV2B. Despite their homology to transporter proteins, the function of the SV2s remains unknown. In an effort to determine SV2 function and identify cofactors required for SV2 activity, we examined the protein interactions of SV2 using a combination of cross-linking, immunoprecipitation, and recombinant protein affinity chromatography. We report that SV2 is part of a large protein complex that contains the synaptic vesicle protein synaptotagmin. The interaction between SV2 and synaptotagmin is direct, specific to SV2A, and inhibited by calcium with an EC50 of approximately 10 microM. Interaction is mediated by the cytoplasmic amino terminus of SV2A and the C2B domain of synaptotagmin. Our observations suggest a regulatory relationship between these two proteins.

Animals↗
PubMed1995

The biochemistry of neurotransmitter secretion.

The progress that has resulted from the convergence of biochemistry with yeast genetics has accelerated the pace at which the molecular events of membrane transport are being elucidated. Future research will focus not only on testing the proposed sequence of protein-protein interactions but also on identifying how calcium regulation is imposed on this system. As our understanding of the basic mechanisms of neurosecretion increases, attention will undoubtedly shift to how the molecules of release are modified to produce changes in synaptic efficacy.

Calcium↗
PubMed1994

Differential expression of synaptic vesicle protein 2 (SV2) isoforms.

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.

Animals↗
PubMed1993

Brain contains two forms of synaptic vesicle protein 2.

Molecular cloning of a cDNA encoding synaptic vesicle protein 2 (SV2) revealed that it is homologous to a family of proton cotransporters from bacteria and fungi and to a related family of glucose transporters found in mammals. The similarity to proton cotransporters raised the possibility that SV2 might mediate the uptake of neurotransmitters into vesicles, an activity known to require a proton gradient. To determine whether SV2 is a member of a family of vesicular proteins, we used the SV2 clone to screen for similar cDNAs in rat brain. We characterized 42 clones, 25 of which encode SV2 and 4 of which encode a protein, SV2B, that is 65% identical and 78% similar to SV2. The protein encoded by SV2B cDNA is recognized by the monoclonal antibody that defines the SV2 protein. When SV2B is expressed in COS cells, antibody labeling is reticular in nature, suggesting that SV2B, like SV2 (hence, SV2A), is segregated to intracellular membranes. The expression of SV2B is limited to neural tissue. While both forms of SV2 are expressed in all brain regions, SV2B is expressed at highest levels in the cortex and hippocampus, whereas the highest level of expression of SV2A is in subcortical regions. Therefore, the SV2 proteins, like other characterized synaptic vesicle proteins, comprise a small gene family.

Amino Acid Sequence↗
PubMed1993

Ceramide 1-phosphate phosphatase activity in brain.

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

Amidohydrolases↗
PubMed1992

SV2, a brain synaptic vesicle protein homologous to bacterial transporters.

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein specifically localized to secretory vesicles in neurons and endocrine cells. As a first step toward understanding the function of SV2 in neural secretion, a rat brain complementary DNA (cDNA) that encodes SV2 was isolated and characterized. Analyses of this cDNA predict that SV2 contains 12 transmembrane domains. The NH2-terminal half of the protein shows significant amino acid sequence identity to a family of bacterial proteins that transport sugars, citrate, and drugs. Expression of the SV2 cDNA in COS cells yielded a high level of SV2-like immunoreactivity distributed in a reticular and punctate pattern, which suggests localization to intracellular membranes. Its localization to vesicles, predicted membrane topology, and sequence identity to known transporters suggest that SV2 is a synaptic vesicle-specific transporter.

Amino Acid Sequence↗
PubMed1989

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles.

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.

Animals↗
PubMed1986

Thyrotropin-releasing hormone stimulation of polyphosphoinositide hydrolysis in GH3 cell membranes is GTP dependent but insensitive to cholera or pertussis toxin.

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).

Animals↗
PubMed1986

Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism.

Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone-stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni.

Adenine Nucleotides↗
PubMed1985

Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism.

Polyphosphoinositide hydrolysis was examined in membranes from thyrotropin-releasing hormone (TRH)-responsive GH3 pituitary cells. [3H]Inositol phosphates (IP2 and IP3) were generated upon incubation of membranes from [3H]inositol-labeled cells indicating the presence of a membrane-associated polyphosphoinositide phosphodiesterase (PPI PDE). Membrane PPI PDE activity was found to be stimulated by TRH and by GTP-gamma-S in Ca2+-modulated manner. In addition, TRH-stimulated PPI hydrolysis was potentiated by GTP. These results demonstrate direct in vitro effects of a hormone on PPI turnover and suggest the involvement of a GTP-binding component in transmembrane signalling by TRH.

Animals↗