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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Epac is an acronym for the exchange proteins activated directly by cyclic AMP, a family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs) that mediate protein kinase A (PKA)-independent signal transduction properties of the second messenger cAMP. Two variants of Epac exist (Epac1 and Epac2), both of which couple cAMP production to the activation of Rap, a small molecular weight GTPase of the Ras family. By activating Rap in an Epac-mediated manner, cAMP influences diverse cellular processes that include integrin-mediated cell adhesion, vascular endothelial cell barrier formation, and cardiac myocyte gap junction formation. Recently, the identification of previously unrecognized physiological processes regulated by Epac has been made possible by the development of Epac-selective cyclic AMP analogues (ESCAs). These cell-permeant analogues of cAMP activate both Epac1 and Epac2, whereas they fail to activate PKA when used at low concentrations.

Ca2؉ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca 2؉ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca 2؉ -regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca 2؉ in cAMP production. We report that depolarization with high external K ؉ , tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca 2؉ and inhibited by nitrendipine, a Ca 2؉ channel blocker, or 2,5-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca 2؉ and cAMP oscillations that were abolished in the absence of extracellular Ca 2؉ and blocked by 2,5-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca 2؉ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca 2؉ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca 2؉ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca 2؉ -dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

channels, and was blocked by treatment with nimodipine, thapsigargin, or ryanodine, but not by the IP 3 receptor antagonist xestospongin C.3. Treatment with the cAMP antagonist 8-Br-Rp-cAMPS blocked CICR in response to exendin-4, whereas the PKA inhibitor H-89 was ineffective when tested at a concentration demonstrated to inhibit PKA-dependent gene expression. RT-PCR of INS-1 cells demonstrated expression of mRNA coding for the type-II isoform of cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF-II, Epac2).5. CICR in response to forskolin was blocked by transient transfection and expression of a dominant negative mutant isoform of cAMP-GEF-II in which inactivating mutations were introduced into the exchange factor's two cAMP-binding domains.6. It is concluded that CICR in INS-1 cells results from GLP-1 receptor-mediated sensitization of the intracellular Ca 2+ release mechanism, a signal transduction pathway independent of PKA, but which requires cAMP-GEF-II.