α-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin Iα was previously described as a high affinity ...α-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of α-latrotoxin with neurexin Iα cannot explain how α-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of α-latrotoxin (CIRL), which is a second high affinity α-latrotoxin receptor that may be the major mediator of α-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 α-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an α-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.
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.
α-Latrotoxin (α-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 α-LTx. Unlike neurexin Ia, a previously described high affinity α-LTx receptor, this novel protein binds α-LTx independently of Ca2+presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by α-latrotoxin. The major protein component of calcium-independent α-LTx receptors is a novel Mr 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the Mr 120,000 protein was found only in brain.
Abstract
Background EphB4 is upregulated in prostate cancer in over half the cases, and correlates with stage and survival. EprhinB2, the ligand for EphB4 has not been well studied. Functional ...studies show EphB4 provides a survival signal through the PI3K/PTEN/Akt/pS6 pathway. A therapeutic agent, soluble EphB4-albumin fusion protein (sEphB4) which blocks bidirectional signaling is currently in clinical development. We thus wished to conduct detailed investigation into the role of EphB4 and EphrinB2 in genetic mouse model of prostate cancer, in tumor initiation and progression, by knockout of EphB4 and by pharmacological agent (sEphB4).
Methods We studied the expression of Ephrinb2 in 180 samples of human prostate cancers and normal prostates using highly characterized, specific EphrinB2 mAb. We next used genetically engineered mouse model of prostate cancer condition deletion of PTEN in prostate epithelium, crossed with transgenic mouse line expressing luciferase. To test the role of EphB4-EphrinB2, we generated Floxed allele of Ephb4 to conditionally delete in prostate epithelium. PTENf/f, EphB4f/f and luciferase were crossed and imaged over time. Secondly, PTENf/f mice were treated with sEphB4 in prevention, established tumor and castration resistant prostate cancer (CRPC) cohorts. Prostate cell lines C4-2B, PC3 and 22RV1 were used to analyze the role of EphB4 in regulation of PI3K pathway and androgen receptor (AR).
Results EphrinB2 is expressed in 50% of human prostate cancers, but not the normal tissues. PTEN null mouse prostate tumors had elevated EphB4 and EphrinB2, but not other family members. Conditional deletion of EphB4 in the context of PTEN deletion in prostate epithelium abolished tumor formation and caused inhibition of the PI3K pathway. Treatment with sEphB4 precluded tumor development and induced tumor regression in both early prostate cancer and CRPC mice. PI3K/AKT/pS6 activity nearly complete declined in the treatment group. Mechanistic studies using EphB4 knock down showed downregulation of PI3K alpha, beta, not gamma and delta. Most surprisingly, AR levels were markedly reduced in sEphB4 treated tumors. Further studies in vitro showed decline in AR with EphB4 knock down, which could be rescued with ectopic expression of PI3K beta, but not other PI3K isoforms. These changes were recapitulated in a patient with late stage CRPC treated in a single patient IND for sEphB4.
Conclusions EphB4-EphrinB2, a receptor-ligand pair are expressed in prostate cancer and induced by loss of PTEN or activation of PI3K pathway. Genetic and pharmacological intervention validates the potential role as a downstream effectors through downregulation of PI3K signaling and AR. Drugs that can reduce AR levels and PI3Kβ inhibitors are potential candidates for prostate cancer therapy and sEphB4 holds therapeutic potential in prostate cancer.
Citation Format: Grace X. Li, Binyun Ma, Valery G. Krasnoperov, Imran Siddiqi, Akash Sali, Gangning Liang, Inderbir S. Gill, Jacek K. Pinski, David I. Quinn, Sarmad Sadeghi, Parkash S. Gill. EphB4-EphrinB2 receptor-ligand are downstream effectors and novel targets of PTEN deficient prostate cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3139.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not
α-Latrotoxin binding to the calcium-independent receptor for α-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 α-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca 2+ . α-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 α-latrotoxin. α-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 α-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. α-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 1α, an
α-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type
CIRL-1. We propose that α-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that α-latrotoxin
itself controls the conductance properties of the channels it produces.
The venom of the black widow spider (BWSV) ( Latrodectus mactans tredecimguttatus ) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific
manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown,
and they have not been functionally expressed. This study reports on the primary structure of -latroinsectotoxin ( -LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. -LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree
of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain
organization of -LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted
molecular mass and apparent mobility of the protein ( 130 kDa) encoded in the -LIT gene differs from that of native -LIT purified from BWSV ( 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native -LIT revealed a molecular ion with m/z + of 110916 ± 100, indicating that the native -LIT is 991 amino acids in length. When the full-length -LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing
991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar
concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native -LIT have a high Ca permeability, whereas those formed by truncated, recombinant protein do not.
α-Latrotoxin (α-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 Ca
...2+
-independent receptor for α-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of α-Ltx on secretion from untransfected cells. α-Ltx, by binding in a Ca
2+
-
independent
manner to an endogenous receptor, causes subsequent Ca
2+
-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca
2+
influx caused by the toxin. In permeabilized cells in which the Ca
2+
concentration is regulated by buffer, α-Ltx also enhances Ca
2+
-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 α-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of α-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 α-Ltx may act by reversing the inhibitory effects of the receptor.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of δ-latroinsectotoxin (δ-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. δ-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of δ-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (~130 kDa) encoded in the δ-LIT gene differs from that of native δ-LIT purified from BWSV (~110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native δ-LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native δ-LIT is 991 amino acids in length. When the full-length δ-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native δ-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
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