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PubMed2001

Intracellular injection of synapsin I induces neurotransmitter release in C1 neurons of Helix pomatia contacting a wrong target.

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I. These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.

Actins↗
PubMed1990

Limited proteolysis of synapsin I. Identification of the region of the molecule responsible for its association with microtubules.

Synapsin I is a highly asymmetric neuronal structural phosphoprotein implicated in the regulation of neurotransmitter release probably by the multiple interactions it can contract with membranous and cytoskeletal elements of the neuronal cell. In order to locate the region(s) of synapsin I responsible for its association with microtubules, we have first studied synapsin I limited digestion by trypsin. The resulting polypeptides were localized in the synapsin I molecule by using three different criteria: their kinetics of appearance, their collagenase sensitivity, and the presence of the synapsin phosphorylation site 1 (cyclic AMP dependent). Synapsin I digestion kinetics are not affected by phosphorylation at this site. Analysis of the ability of various synapsin I tryptic fragments in mixture to cosediment with microtubules shows that a 44-kDa fragment corresponding to the NH2-terminal hydrophobic head of the molecule contains a binding site for polymerized tubulin. This fragment competes with native synapsin I for binding on microtubules. None of the polypeptides belonging to the tail region of synapsin I (COOH-terminal half of the molecule) were found to cosediment with microtubules.

Animals↗
PubMedDate not supplied

Synapsin I is a microtubule-bundling protein.

Synapsin I, a synaptic vesicle protein, is thought to be involved in the regulation of neurotransmission through its phosphorylation by the cyclic AMP-dependent and Ca2+/calmodulin-dependent protein kinases which become activated upon depolarization of nerve endings. However, despite its recent characterization as a spectrin-binding protein immunologically related to erythrocyte protein 4.1, other interactions of synapsin I with structural proteins remain unknown. We report here that synapsin I can co-cycle with microtubules through three cycles of warm polymerization and cold depolymerization. Synapsin I binds saturably to microtubules stabilized by taxol, with an estimated dissociation constant (Kd) of 4.5 microM and a stoichiometry of 1.2 mol of synapsin binding sites per mol tubulin dimer. Synapsin I also increases the turbidity of tubulin solutions at 37 degrees C, but without causing detectable alterations in the critical concentration required for polymerization. Mixtures of synapsin I and tubulin observed by negative stain electron microscopy contain bundles of microtubules, accounting for the effect of synapsin I on tubulin turbidity. Synapsin I is thus a candidate to mediate or regulate the interaction of synaptic vesicles with microtubules.

Alkaloids↗
PubMed2001

Identification of synapsin I peptides that insert into lipid membranes.

The synapsins constitute a family of synaptic vesicle-associated phosphoproteins essential for regulating neurotransmitter release and synaptogenesis. The molecular mechanisms underlying the selective targeting of synapsin I to synaptic vesicles are thought to involve specific protein-protein interactions, while the high-affinity binding to the synaptic vesicle membrane may involve both protein-protein and protein-lipid interactions. The highly hydrophobic N-terminal region of the protein has been shown to bind with high affinity to the acidic phospholipids phosphatidylserine and phosphatidylinositol and to penetrate the hydrophobic core of the lipid bilayer. To precisely identify the domains of synapsin I which mediate the interaction with lipids, synapsin I was bound to liposomes containing the membrane-directed carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and subjected to photolysis. Isolation and N-terminal amino acid sequencing of 125I-labelled synapsin I peptides derived from CNBr cleavage indicated that three distinct regions in the highly conserved domain C of synapsin I insert into the hydrophobic core of the phospholipid bilayer. The boundaries of the regions encompass residues 166-192, 233-258 and 278-327 of bovine synapsin I. These regions are surface-exposed in the crystal structure of domain C of bovine synapsin I and are evolutionarily conserved among isoforms across species. The present data offer a molecular explanation for the high-affinity binding of synapsin I to phospholipid bilayers and synaptic vesicles.

Amino Acid Sequence↗
PubMed1991

Site specificity in the interactions of synapsin 1 with tubulin.

Synapsin 1 is one of a family of phosphoproteins located on small synaptic vesicles (SSV) in the presynaptic terminal, and probably plays a critical role in the process of neuronal exocytosis by providing regulated linkages between SSV and the cytoskeleton. Two forms of synapsin 1 are produced from a single gene by differential mRNA splicing: 1a, 706 amino acid residues, and 1b, 670 residues. Synapsin 1 has two structural domains, a globular N-terminal head domain and an elongated tail domain. Electron microscopy of nerve terminals in situ and reconstitution studies in vitro indicates that synapsin 1 can interact with microtubules, microfilaments and brain spectrin. In vitro, synapsin 1 can bundle microtubules. This could either occur by synapsin 1 being at least bivalent for microtubules, or by univalent synapsin 1 molecules aggregating to form complexes that are more than univalent. To resolve this question, we have taken the approach of preparing defined fragments of synapsin 1 from each structural domain and analysing them for tubulin-binding activity. Our results show that there are tubulin-binding sites in both head and tail domains. We conclude that synapsin 1 monomers should be able to cross-link microtubules.

Amino Acid Sequence↗
PubMed1994

Synapsin IIa bundles actin filaments.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

Actins↗
PubMed2000

Cyclosporine A-induced hypertension involves synapsin in renal sensory nerve endings.

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.

Afferent Pathways↗
PubMed1991

Cytosolic rat brain synapsin I is a diacylglycerol kinase.

The phosphorylation of diacylglycerol (DG), a reaction catalyzed by DG kinase, may be critical in the termination of effector-induced signals mediated by protein kinase C. Synapsin I is a principal target of intracellular protein kinases and is thought to be involved in the release of neurotransmitter from axon terminals. We present several lines of evidence which indicate that rat brain synapsin, in addition to this role, may function as a DG kinase. Purified rat brain DG kinase was digested with trypsin, which produced three major fragments whose sequence was identical to three regions in synapsin I. Using a rabbit anti-synapsin polyclonal antiserum, the elution profile of synapsin immunoreactivity coincided exactly with that of DG kinase activity in column fractions from the final step in the DG kinase purification procedure. As is the case with synapsin, the purified enzyme was a strongly basic protein with an isoelectric point greater than 10.0. Finally, incubating the DG kinase with highly purified bacterial collagenase, an enzyme that partially degrades the proline- and glycine-rich synapsin, resulted in the simultaneous loss of DG kinase activity and synapsin immunoreactivity. We conclude that cytosolic rat brain synapsin is capable of functioning as a DG kinase.

Amino Acid Sequence↗
PubMed1997

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity.

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

Animals↗
PubMed2003

Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I.

Synapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Galphaz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I(-/-)/II(-/-) mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery.

Alternative Splicing↗
PubMed1988

Multiple synapsin I messenger RNAs are differentially regulated during neuronal development.

Synapsin I is a neuron-specific protein consisting of two isoforms Ia and Ib. It is thought to play a role in the regulation of neurotransmitter release. In this study the structure and expression of two classes of synapsin I mRNA have been examined. The two mRNA classes have molecular sizes of 3.4 and 4.5 kb, respectively. Both classes translate into synapsin I polypeptides and display a high degree of base sequence homology. Utilizing an oligonucleotide-directed RNase H assay we have shown that both mRNA classes have a common start site of transcription and differ from one another toward their 3' ends. The expression of the two synapsin I mRNA classes is differentially regulated during the development of the rat brain and cerebellum. In the cerebellum the 4.5-kb transcript is expressed until postnatal day 7, after which it decreases to an undetectable level. The 3.4-kb mRNA is found throughout cerebellar development and in the adult. This suggests that the 3.4-kb mRNA class consists of messages which can encode both synapsin I polypeptides. Using quantitative Northern blot analysis a peak in the expression of this mRNA was observed at postnatal day 20. The maximum expression of the 3.4-kb class coincides with the period of synaptogenesis in the cerebellum. In addition to the developmental time course of synapsin mRNA expression a description of its spatial distribution throughout the cerebellum was performed using in situ hybridization histochemistry. From postnatal day 15 onwards, with a maximum at postnatal day 20, synapsin mRNA was localized in the internal granule cell layer of the cerebellum. On a cellular level, the granule cells, but not the neighboring Purkinje cells, express high levels of synapsin mRNA. These observations implicate developmentally coordinated differential RNA splicing in the regulation of neuron-specific gene expression and substantiate the correlation of synapsin gene expression with the period of synaptogenic differentiation of neurons.

Animals↗
PubMed1989

Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin.

Synapsin I is a neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles. It has been demonstrated to bundle F-actin in a phosphorylation-dependent manner in vitro, a property consistent with its proposed role in linking synaptic vesicles to the cytoskeleton and its involvement in the regulation of neurotransmitter release. Synapsin I is composed of two distinct domains, a COOH terminal, collagenase-sensitive, hydrophilic, and strongly basic tail region, and an NH2 terminal, collagenase-resistant head region relatively rich in hydrophobic amino acids. To elucidate the structural basis for the interactions between synapsin I and F-actin and how it relates to other characteristics of synapsin I, we have performed a structure-function analysis of fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid. The fragments were identified and aligned with the parent molecule using the deduced primary structure of synapsin I and the known phosphorylation sites as markers. We have purified these fragments and examined their interactions with F-actin. Two distinct fragments, a 29-kD NH2-terminal fragment and a 15-kD middle fragment, were shown to contain F-actin binding sites. A 51/54-kD middle/tail fragment retained the F-actin binding and bundling activity of synapsin I, but the isolated tail fragment did not retain either activity. In contrast to phosphorylation of sites two and three in intact synapsin I, which abolishes F-actin bundling activity, phosphorylation of these sites in the middle/tail fragment failed to abolish this activity. In conclusion, three domains of synapsin I appear to be involved in F-actin binding and bundling.

Actins↗
PubMed1987

Structural and functional relationship of red blood cell protein 4.1 to synapsin I.

It has been suggested that the neuron specific protein synapsin I is closely related to red blood cell (rbc) protein 4.1. A systematic comparison of the structural and functional properties of rbc protein 4.1 and synapsin I has been carried out. There is approximately a three order of magnitude difference in cross reactivity of synapsin I with rbc 4.1 antiserum vs. synapsin I antiserum, as determined by a competitive quantitative dot assay. Two-dimensional chymotryptic iodopeptide mapping analysis demonstrated limited peptide homology (approximately 34% spot overlap) between rbc 4.1 and synapsin I. Dephosphorylated synapsin I binds saturably to brain spectrin (240/235) with an estimated dissociation constant (Kd) of 700 nM and a maximal binding capacity of 4 mol synapsin I/mol spectrin tetramer, similar to the affinity and stoichiometry of 4.1 binding to rbc spectrin. Synapsin I was found to bind to the terminal ends of the brain spectrin tetramer by low-angle rotary shadowing, analogous to 4.1 binding to rbc spectrin. In summary, synapsin I is structurally and immunologically distinct from rbc 4.1, yet shares functional similarities with rbc 4.1 with respect to its spectrin binding characteristics.

Animals↗
PubMed2005

Structural domains involved in the regulation of transmitter release by synapsins.

Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structure-function analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.

Amino Acid Sequence↗
PubMed1999

Molecular evolution of the synapsin gene family.

Synapsins, a family of synaptic vesicle proteins, play a crucial role in the regulation of neurotransmission and synaptogenesis. They have been identified in a variety of invertebrate and vertebrate species, including human, rat (Rattus norvegicus), cow (Bos taurus), longfin squid (Loligo pealei), and fruit fly (Drosophila melanogaster). Here, synapsins were cloned from three additional species: frog (Xenopus laevis), lamprey (Lampetra fluviatilis), and nematode (Caenorhabditis elegans). Synapsin protein sequences from all these species were then used to explore the molecular phylogeny of these important neuronal phosphoproteins. The ancestral condition of a single synapsin gene probably gave rise to the vertebrate synapsin gene family comprised of at least three synapsin genes (I, II, and III) in higher vertebrates. Synapsins possess multiple domains, which have evolved at different rates throughout evolution. In invertebrate synapsins, the most conserved domains are C and E. During the evolution of vertebrates, at least two gene duplication events are hypothesized to have given rise to the synapsin gene family. This was accompanied by the emergence of an additional conserved domain, termed A. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:360-377, 1999.

Amino Acid Sequence↗
PubMed1992

Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.

Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.

Actins↗
PubMed1990

Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes.

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II. The steady-state emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the long-living chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylated form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.

2-Naphthylamine↗
PubMed1987

Synapsin I in PC12 cells. I. Characterization of the phosphoprotein and effect of chronic NGF treatment.

PC12 cells contain a synapsin I-like molecule. Several serum and monoclonal antibodies raised against bovine brain synapsin I bind to and precipitate this molecule, demonstrating immunochemical similarity between the brain and PC12 species. PC12 synapsin I, like brain synapsin I, is a phosphoprotein: It is phosphorylated in intact cells and, when partially purified, serves as a substrate for several synapsin I kinases. PC12 cell synapsin I is structurally similar to brain synapsin I as shown by peptide mapping of 35S-methionine-and 32P-phosphate-labeled molecules from the 2 sources. Chronic NGF treatment of the cells induces a significant increase in the amount of synapsin I relative to total cell protein, measured either by immunolabeling or incorporation of 35S-methionine. The synapsin I present in untreated PC12 cells migrates predominantly as a singlet and that present in cells treated chronically with NGF as a doublet in SDS-PAGE.

Adrenal Gland Neoplasms↗