[Systemic approach to clinical laboratory diagnosis of infectious endocarditis].
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Biomedical subjects
A G Petrenko
Publications and source records attributed to A G Petrenko.
Calcium-independent receptor for alpha-latrotoxin and neurexin 1alpha [corrected] facilitate toxin-induced channel formation: evidence that channel formation results from tethering of toxin to membrane.
alpha-Latrotoxin binding to the calcium-independent receptor for alpha-latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of alpha-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca(2+). alpha-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca(2+)-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by alpha-latrotoxin. alpha-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca(2+). At alpha-latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. alpha-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1alpha, an alpha-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that alpha-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that alpha-latrotoxin itself controls the conductance properties of the channels it produces.
Animals↗
A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors.
Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.
Amino Acid Sequence↗
Structural requirements for alpha-latrotoxin binding and alpha-latrotoxin-stimulated secretion. A study with calcium-independent receptor of alpha-latrotoxin (CIRL) deletion mutants.
Stimulation of neurotransmitter release by alpha-latrotoxin requires its binding to the calcium-independent receptor of alpha-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-latrotoxin-induced calcium uptake in HEK293 cells and alpha-latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-latrotoxin.
Animals↗
A Ca2+-independent receptor for alpha-latrotoxin, CIRL, mediates effects on secretion via multiple mechanisms.
alpha-Latrotoxin (alpha-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for alpha-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of alpha-Ltx on secretion from untransfected cells. alpha-Ltx, by binding in a Ca2+-independent manner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+ influx caused by the toxin. In permeabilized cells in which the Ca2+ concentration is regulated by buffer, alpha-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of alpha-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of alpha-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that alpha-Ltx may act by reversing the inhibitory effects of the receptor.
Adenosine Triphosphate↗
Neurexin I alpha is a major alpha-latrotoxin receptor that cooperates in alpha-latrotoxin action.
alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities; Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. We have now generated and analyzed mice that lack neurexin I alpha to test its importance in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice was decreased by almost 50% compared with wild type membranes; the decrease was almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin was still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, showed a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, our results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin.
Alternative Splicing↗
alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor.
alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.
Amino Acid Sequence↗
The calcium-independent receptor of alpha-latrotoxin is not a neurexin.
alpha-Latrotoxin (alpha-LTx), a vertebrate neurotoxin isolated from Black Widow Spider venom, causes massive spontaneous neurotransmitter release. The molecular mechanism(s) by which the toxin exerts its effect is largely unknown. Here we report identification and purification of a novel membrane receptor with high affinity for alpha-LTx. Unlike neurexin Ia, a previously described high affinity alpha-LTx receptor, this novel protein binds alpha-LTx independently of Ca2+ presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by alpha-latrotoxin. The major protein component of calcium-independent alpha-LTx receptors is a novel M(r) 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the M(r) 120,000 protein was found only in brain.
Amino Acid Sequence↗
Structure and evolution of neurexophilin.
Using affinity chromatography on immobilized alpha-latrotoxin, we have purified a novel 29 kDa protein, neurexophilin, in a complex with neurexin l alpha. Cloning revealed that rat and bovine neurexophilins are composed of N-terminal signal peptides, nonconserved N-terminal domains (20% identity over 80 residues), and highly homologous C-terminal sequences (85% identity over 169 residues). Analysis of genomic clones from mice identified two distinct neurexophilin genes, one of which is more homologous to rat neurexophilin and the other to bovine neurexophilin. The first neurexophilin gene is expressed abundantly in adult rat and mouse brain, whereas no mRNA corresponding to the second gene was detected in rodents despite its abundant expression in bovine brain, suggesting that rodents and cattle primarily express distinct neurexophilin genes. RNA blots and in situ hybridizations revealed that neurexophilin is expressed in adult rat brain at high levels only in a scattered subpopulation of neurons that probably represent inhibitory interneurons; by contrast, neurexins are expressed in all neurons. Neurexophilin contains a signal sequence and is N-glycosylated at multiple sites, suggesting that it is secreted and binds to the extracellular domain of neurexin l alpha. This hypothesis was confirmed by binding recombinant neurexophilin to the extracellular domains of neurexin l alpha. Together our data suggest that neurexophilin constitutes a secreted glycoprotein that is synthesized in a subclass of neurons and may be a ligand for neurexins.
Amino Acid Sequence↗
High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha.
alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I alpha binds alpha-latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). Our data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin.
Alternative Splicing↗
alpha-Latrotoxin receptor. Implications in nerve terminal function.
alpha-Latrotoxin is a potent stimulator of neurotransmitter release from nerve terminals. High affinity membrane alpha-latrotoxin receptor was purified in an active binding form. It is a membrane glycoprotein (M(r) 160,000-220,000) which may be complexed to a smaller polypeptide (M(r) 29,000). The structure of the receptor protein suggests that it may be a synapse-specific cell recognition molecule. Intracellularly, the alpha-latrotoxin receptor interacts with synaptotagmin, a calcium- and phospholipid-binding protein specifically localized in the synaptic vesicle membrane. This interaction may be important for targeting of synaptic vesicles to presynaptic release sites.
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Phosphorylation of synaptotagmin I by casein kinase II.
Synaptotagmin I is an abundant synaptic vesicle protein that binds Ca2+ in a phospholipid-dependent manner and is thought to function in synaptic vesicle exocytosis. We have now studied the phosphorylation of synaptotagmin I. Synaptotagmin I is one of the major substrates in brain for casein kinase II, which phosphorylates synaptotagmin at a single threonine. The phosphorylation site was mapped using recombinant proteins to threonine 128 of synaptotagmin I, which is located in the sequence between the transmembrane region and the C2 domain repeats of synaptotagmin I. The phosphorylation site of synaptotagmin I is also present in synaptotagmin II and is evolutionarily conserved between different species. Preceding the phosphorylation site, synaptotagmins I and II contain a lysine-rich sequence. Casein kinase II phosphorylation of many substrates is strongly stimulated by the addition of polylysine, but phosphorylation of synaptotagmin I by casein kinase II is not. In recombinant proteins, removal of the lysine-rich sequence of synaptotagmin I makes its phosphorylation dependent on exogenous polylysine, suggesting that the lysine-rich sequence in synaptotagmin serves as an endogenous polylysine stimulation signal for casein kinase II. Our data demonstrate that synaptotagmin I is an efficient substrate for casein kinase II at a conserved site with a possible modulatory role in nerve terminal function.
Amino Acid Sequence↗
Polypeptide composition of the alpha-latrotoxin receptor. High affinity binding protein consists of a family of related high molecular weight polypeptides complexed to a low molecular weight protein.
alpha-Latrotoxin is a vertebrate neurotoxin from black widow spider venom that causes massive neurotransmitter release. In order to gain insight into the mechanism of action of alpha-latrotoxin, we have studied alpha-latrotoxin-binding proteins from bovine and rat brain. Proteins purified by affinity chromatography on immobilized alpha-latrotoxin were investigated. Two sets of proteins were isolated: 1) three polypeptides of M(r) 79,000, 65,000, and 43,000 that were eluted from immobilized alpha-latrotoxin by increasing KCl concentrations in the presence of Ca2+, and 2) a family of related proteins ranging in molecular weight from 160,000 to 220,000 and a low molecular weight component of M(r) 29,000 that were eluted from immobilized alpha-latrotoxin only after removal of Ca2+. Amino acid sequences of these proteins demonstrated that all of these proteins represent novel proteins except for the M(r) 65,000 polypeptide, which is identical with synaptotagmin (Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Südhof, T. C. (1991) Nature 353, 65-68). Surprisingly, the M(r) 79,000 and 43,000 proteins were also found in tissues insensitive to alpha-latrotoxin action. Since these proteins do not bind 125I-alpha-latrotoxin with high affinity, their purification probably is not physiologically significant. On the other hand, the fractions containing the M(r) 160,000-220,000 and 29,000 polypeptides bound alpha-latrotoxin with high affinity. Sucrose gradient centrifugations and anion exchange chromatography suggested that most of the M(r) 160,000-220,000 proteins were complexed with the M(r) 29,000 protein. alpha-Latrotoxin binding correlated with the presence of the M(r) 160,000-220,000 proteins and M(r) 29,000 polypeptide, and alpha-latrotoxin formed stable complexes with the M(r) 160,000-220,000 proteins. Accordingly, the alpha-latrotoxin receptor consists of a high molecular weight protein (M(r) 160,000-220,000) that is complexed with one or several copies of an M(r) 29,000 polypeptide. In addition, the receptor is found in a less tight association with synaptotagmin but not with other polypeptides.
Amino Acid Sequence↗
Interaction of synaptotagmin with the cytoplasmic domains of neurexins.
Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin revealed binding of alpha- and beta-neurexins to synaptotagmin in a Ca(2+)-independent manner. Using a series of recombinant proteins in which glutathione S-transferase was fused to the cytoplasmic domains of three different neurexins or of control proteins, it was found that synaptotagmin specifically interacts with the cytoplasmic domains of neurexins but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. Our data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (the neurexins) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal.
Amino Acid Sequence↗
Molecular approaches to synaptic vesicle exocytosis.
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Amino Acid Sequence↗
[Pharmaceutical protection of brain in ischemia and reperfusion experiments].
The results of the use of medicamentous protection of the brain under conditions of modelled ischemia and reperfusion by means of KS-79 preparation are presented. In exclusion of blood flow through the carotid arteries for the period commensurate to that in the clinic, it was established that the use of KS-79 preparation possessing the anti-ischemic and antioxidant properties has pronounced cerebro-prospective action, preserves functional and structural integrity of the brain.
Animals↗
Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin.
A family of highly polymorphic neuronal cell surface proteins, the neurexins, has been identified. At least two genes for neurexins exist. Each gene uses alternative promoters and multiple variably spliced exons to potentially generate more than a 100 different neurexin transcripts. The neurexins were discovered by the identification of one member of the family as the receptor for alpha-latrotoxin. This toxin is a component of the venom from black widow spiders; it binds to presynaptic nerve terminals and triggers massive neurotransmitter release. Neurexins contain single transmembrane regions and extracellular domains with repeated sequences similar to sequences in laminin A, slit, and agrin, proteins that have been implicated in axon guidance and synaptogenesis. An antibody to neurexin I showed highly concentrated immunoreactivity at the synapse. The polymorphic structure of the neurexins, their neural localization, and their sequence similarity to proteins associated with neurogenesis suggest a function as cell recognition molecules in the nerve terminal.
Amino Acid Isomerases↗
Synaptotagmin: a calcium sensor on the synaptic vesicle surface.
Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic vesicles. However, the molecular steps transducing the calcium signal into membrane fusion are still an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle protein, binds calcium at physiological concentrations in a complex with negatively charged phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of synaptotagmin (it is abolished by proteolytic cleavage at a single site). These results suggest that synaptotagmin acts as a cooperative calcium receptor in exocytosis.
Animals↗