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.

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EphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the erbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. The aberrantly expressed receptor was phosphorylated by its natural ligand, EphrinB2, and signaled via the protein kinase B pathway. Targeted knockdown of EphB4 expression by small interference RNA (and antisense oligodeoxynucleotides (ODNs)) led to dose-dependent reduction in cell survival, increased apoptosis, and sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Antisense ODN-mediated EphB4 knockdown resulted in reduced tumor growth in a murine tumor xenograft model. Antisense ODNtreated tumors were 72% smaller than control tumors at 6 weeks, with an 86% reduction in proliferating cells, 15-fold increase in apoptosis, and 44% reduction in tumor microvasculature. Our data indicate that biologically active EphB4 functions as a survival factor in breast cancer and is a novel target for therapy. (Am J