Oleg G. Chepurny

α-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-latrotoxins 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.

Cell physiology of cAMP sensor Epac.

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. ESCAs such as 8‐pCPT‐2′‐O‐Me‐cAMP and 8‐pMeOPT‐2′‐O‐Me‐cAMP are reported to alter Na+, K+, Ca2+ and Cl− channel function, intracellular [Ca2+], and Na+–H+ transporter activity in multiple cell types. Moreover, new studies examining the actions of ESCAs on neurons, pancreatic beta cells, pituitary cells and sperm demonstrate a major role for Epac in the stimulation of exocytosis by cAMP. This topical review provides an update concerning novel PKA‐independent features of cAMP signal transduction that are likely to be Epac‐mediated. Emphasized is the emerging role of Epac in the cAMP‐dependent regulation of ion channel function, intracellular Ca2+ signalling, ion transporter activity and exocytosis.

cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic β-cells

1 The signal transduction pathway responsible for cAMP‐dependent Ca2+‐induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores was assessed in the insulin‐secreting cell line INS‐1. 2 CICR was triggered by the GLP‐1 receptor agonist exendin‐4, an effect mimicked by caffeine, Sp‐cAMPS or forskolin. CICR required influx of Ca2+ through L‐type voltage‐dependent Ca2+ channels, and was blocked by treatment with nimodipine, thapsigargin, or ryanodine, but not by the IP3 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. 4 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 factors two cAMP‐binding domains. 6 It is concluded that CICR in INS‐1 cells results from GLP‐1 receptor‐mediated sensitization of the intracellular Ca2+ release mechanism, a signal transduction pathway independent of PKA, but which requires cAMP‐GEF‐II.

Glucagon-like peptide-1 synthetic analogs: new therapeutic agents for use in the treatment of diabetes mellitus.

Glucagon-like peptide-1-(7-36)-amide (GLP-1) is a potent blood glucose-lowering hormone now under investigation for use as a therapeutic agent in the treatment of type 2 (adult onset) diabetes mellitus. GLP-1 binds with high affinity to G protein-coupled receptors (GPCRs) located on pancreatic beta-cells, and it exerts insulinotropic actions that include the stimulation of insulin gene transcription, insulin biosynthesis, and insulin secretion. The beneficial therapeutic action of GLP-1 also includes its ability to act as a growth factor, stimulating formation of new pancreatic islets (neogenesis) while slowing beta-cell death (apoptosis). GLP-1 belongs to a large family of structurally-related hormones and neuropeptides that include glucagon, secretin, GIP, PACAP, and VIP. Biosynthesis of GLP-1 occurs in the enteroendocrine L-cells of the distal intestine, and the release of GLP-1 into the systemic circulation accompanies ingestion of a meal. Although GLP-1 is inactivated rapidly by dipeptidyl peptidase IV (DDP-IV), synthetic analogs of GLP-1 exist, and efforts have been directed at engineering these peptides so that they are resistant to enzymatic hydrolysis. Additional modifications of GLP-1 incorporate fatty acylation and drug affinity complex (DAC) technology to improve serum albumin binding, thereby slowing renal clearance of the peptides. NN2211, LY315902, LY307161, and CJC-1131 are GLP-1 synthetic analogs that reproduce many of the biological actions of GLP-1, but with a prolonged duration of action. AC2993 (Exendin-4) is a naturally occurring peptide isolated from the lizard Heloderma, and it acts as a high affinity agonist at the GLP-1 receptor. This review summarizes structural features and signal transduction properties of GLP-1 and its cognate beta-cell GPCR. The usefulness of synthetic GLP-1 analogs as blood glucose-lowering agents is discussed, and the applicability of GLP-1 as a therapeutic agent for treatment of type 2 diabetes is highlighted.

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic β cells and rat INS-1 cells

The Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic β cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide‐binding fold‐1 (NBF‐1) of the β‐cell sulphonylurea receptor‐1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit β‐cell ATP‐sensitive K+ channels (KATP channels; a hetero‐octomer of SUR1 and Kir6.2). If so, Epac‐mediated inhibition of KATP channels might explain prior reports that cAMP‐elevating agents promote β‐cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac‐selective cAMP analogues (2′‐O‐Me‐cAMP; 8‐pCPT‐2′‐O‐Me‐cAMP; 8‐pMeOPT‐2′‐O‐Me‐cAMP), but not a cGMP analogue (2′‐O‐Me‐cGMP), inhibit the function of KATP channels in human β cells and rat INS‐1 insulin‐secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6‐Bnz‐cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac‐selective cAMP analogues at KATP channels are mimicked by a cAMP agonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Sp‐isomer, Sp‐8‐Br‐cAMPS), but not a cAMP antagonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer, Rp‐8‐Br‐cAMPS), and are abrogated following transfection of INS‐1 cells with a dominant‐negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full‐length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic β cells.

A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic β cells

The blood glucose‐lowering hormone glucagon‐like peptide‐1 (GLP‐1) stimulates cAMP production, promotes Ca2+ influx, and mobilizes an intracellular source of Ca2+ in pancreatic β cells. Here we provide evidence that these actions of GLP‐1 are functionally related: they reflect a process of Ca2+‐induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin‐secreting INS‐1 cells or mouse β cells loaded with caged Ca2+ (NP‐EGTA), a GLP‐1 receptor agonist (exendin‐4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin‐4 is diminished by an inhibitor of PKA (H‐89) or by overexpression of dominant negative Epac. It is reproduced by cell‐permeant cAMP analogues that activate PKA (6‐Bnz‐cAMP) or Epac (8‐pCPT‐2′‐O‐Me‐cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP‐elevating agents, it is concluded that there exists in β cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels (ryanodine receptors, inositol 1,4,5‐trisphosphate (IP3) receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP‐1 interacts with β cell glucose metabolism to stimulate insulin secretion.

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors.

Poisoning with α-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 α-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 α-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 α-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of α-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of α-latrotoxin, cells of other tissues are also susceptible to the toxic effects of α-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

Isoform-specific antagonists of exchange proteins directly activated by cAMP.

The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC- and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.

Molecular physiology of glucagon-like peptide-1 insulin secretagogue action in pancreatic β cells

Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2-mediated actions of GLP-1 to regulate ATP-sensitive K⁺ channels, voltage-dependent K⁺ channels, TRPM2 cation channels, intracellular Ca⁺ release channels, and Ca⁺-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM.

Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic β-cells and rat INS-1 cells

Protein kinase A (PKA)‐independent actions of adenosine 3′,5′‐cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic β‐cells. Evidence that Epac might mediate the cAMP‐dependent inhibition of β‐cell ATP‐sensitive K+ channels (KATP) was provided by one prior study of human β‐cells and a rat insulin‐secreting cell line (INS‐1 cells) in which it was demonstrated that an Epac‐selective cAMP analogue (ESCA) inhibited a sulphonylurea‐sensitive K+ current measured under conditions of whole‐cell recording. Using excised patches of plasma membrane derived from human β‐cells and rat INS‐1 cells, we now report that 2′‐O‐Me‐cAMP, an ESCA that activates Epac but not PKA, sensitizes single KATP channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2′‐O‐Me‐cAMP (50 μm), the dose–response relationship describing ATP‐dependent inhibition of KATP channel activity (NPo) is left‐shifted such that the concentration of ATP producing 50% inhibition (IC50) is reduced from 22 μm to 1 μm for human β‐cells, and from 14 μm to 4 μm for rat INS‐1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 μm), the administration of 2′‐O‐Me‐cAMP inhibits channel activity in a dose‐dependent and reversible manner (IC50 12 μm for both cell types). A cyclic nucleotide phosphodiesterase‐resistant ESCA (Sp‐8‐pCPT‐2′‐O‐Me‐cAMPS) also inhibits KATP channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human β‐cells and rat INS‐1 cells a novel form of ion channel modulation in which the ATP sensitivity of KATP channels is regulated by Epac.