Cell physiology of cAMP sensor Epac

Abstract

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(+), Ca(2+) and Cl(-) channel function, intracellular [Ca(2+)], 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 Ca(2+) signalling, ion transporter activity and exocytosis.

Figure 1. Signal transduction properties of Epac

When bound to cAMP, Epac catalyses the exchange of GDP for GTP on Rap GTPase. The activated form of Rap-GTP is then capable of promoting integrin-mediated cell adhesion, gap junction formation and ERK1/2 MAPK-mediated protein phosphorylation. Activated Rap also stimulates phospholipase C-epsilon (PLC-ɛ) which hydrolyses PIP₂ to generate diacylglycerol (DAG), and the Ca²⁺-mobilizing second messenger IP₃. As illustrated, some actions of Epac may also be Rap independent. These actions of Epac may involve its interaction with microtubule-associated proteins, the Ras GTPases, secretory granule-associated proteins (Rim2, Piccolo), and the SUR1 subunit of K_ATP channels. Abbreviations: G protein coupled receptor, GPCR; cytoskeletal protein-associated with the active zone, CAZ; ATP-sensitive K⁺ channel, K_ATP.

Figure 2. Molecular properties of the Epac family of cAMPGEFs

Epac1 is comprised of 881 amino acids (molecular mass 100 kDa), whereas Epac2 is comprised of 1011 amino acids (molecular mass 110 kDa). In the absence of cAMP, the regulatory region of Epac inhibits the guanine nucleotide exchange (GEF) function of the catalytic region. Binding of cAMP to Epac relieves this autoinhibition. The DEP domain of Epac located within the regulatory region contains sequence homologies to disheveled, Egl I0 and pleckstrin. A Ras exchange motif (REM) and a CDC25 homology domain are found within the catalytic region. These two variants of Epac are coded for by two distinct genes, and evidence exists for both shorter and longer forms of the proteins (not shown).

Figure 3. Detection of [cAMP]_i using Epac1-camps

The [cAMP]_i was measured in a single MIN6 insulin-secreting cell transfected with Epac1-camps, a cAMP sensor that incorporates the cyclic nucleotide-binding domain of Epac1 fused at its C-terminus with ECFP (FRET donor), and at its N-terminus with EYFP (FRET acceptor). Emitted light was measured at 485 and 535 nm in response to excitation at 440 nm (Landa et al. 2005). An increase of [cAMP]_i produces a decrease of FRET. This action of cAMP is measured as a decrease of 535 nm emitted light accompanied by an increase of 485 nm emitted light. A, a MIN6 cell equilibrated in saline containing 2 mm glucose, and then challenged with a solution containing 20 mm glucose with or without 20 mm of the K⁺ channel blocker tetraethylammonium ion (TEA). Application of 20 mm glucose alone produced a small increase of [cAMP]_i, whereas larger oscillations of [cAMP]_i were observed upon introduction of TEA to the bath solution. TEA initiates oscillatory electrical activity in this cell type, an effect accompanied by oscillations of both [Ca²⁺]_i and [cAMP]_i (Landa et al. 2005). B, data presented in panel A re-plotted as the relative ratio of 485/535 nm emitted light versus time. The complete experiment encapsulating 2160 s is illustrated.

Figure 4. Chemical structures of cAMP analogues

Illustrated are the structures for cAMP (A), 8-pCPT-2′-O-Me-cAMP (B), 8-pMeOPT-2′-O-Me-cAMP (C), and 6-Bnz-cAMP (D). Both 8-pCPT-2′-O-Me-cAMP and 8-pMeOPT-2′-O-Me-cAMP are Epac selective, whereas 6-Bnz-cAMP is PKA selective. The naturally occurring second messenger cAMP activates both Epac and PKA. A chlorophenylthio substitution introduced at the 8′ position of cAMP (B and C) dramatically increase the lipophilicity of the cAMP analogues, thereby rendering them cell-permeant. Although not shown, an Sp- isomer of 8-pCPT-2′-O-Me-cAMP is also available. It activates Epac, whereas the Rp- isomer does not.

Figure 5. cAMP may inhibit K_ATP channel function in an Epac-mediated manner

Nucleotide-binding fold-1 (NBF-1) of the SUR1 subunit of K_ATP channels may recruit Epac to the plasma membrane. Binding of cAMP to Epac may then allow for the activation of plasma membrane-associated Rap GTPase. The activated form of Rap stimulates PLC-ɛ, and the PLC-ɛ-catalysed hydrolysis of PIP₂ results in the closure of K_ATP channels, possibly as a consequence of the increased sensitivity of these channels to ATP. Note that ATP inhibits K_ATP channel function by virtue of its interaction with the Kir6.2 subunit of the channel. In contrast, the activity of K_ATP channels is stimulated by Mg²⁺-ADP, acting at the SUR1 subunit. Abbreviations: W_A and W_B, Walker A and Walker B motifs; TMO, TM1 and TM2, transmembrane clusters; NBF-2, nucleotide-binding fold-2.

Figure 6. Epac mediates the Ca²⁺-mobilizing action of cAMP

The mobilization of Ca²⁺ from endoplasmic reticulum (ER) Ca²⁺ stores may be facilitated as a consequence of the Epac-mediated action of cAMP to promote the opening of intracellular Ca²⁺ release channels corresponding to inositol trisphosphate receptors (IP₃-R) or ryanodine receptors (RYR). Such an effect of cAMP might be explained by the ability of Epac to interact directly with the channels. A second possibility is that Epac acts via Rap GTPases to stimulate protein kinases that phosphorylate and regulate the function of intracellular Ca²⁺ release channels. A third possibility is that the Epac-mediated activation of Rap GTPases leads to the stimulation of PLC-ɛ, which generates IP₃ by hydrolysing PIP₂. Abbreviations: GPCR, G protein-coupled receptor; SERCA, sarco-endoplasmic reticulum ATPase.

Figure 7. Interactions of Epac2 with secretory granule-associated proteins

A, in the model of Seino and co-workers, plasma membrane SUR1 (pmSUR1), Epac2, Rim2 and Piccolo form a macromolecular complex that interacts with the GTP-bound form of Rab3A to regulate the priming and exocytosis of secretory granules (SG). This model may also apply to presynaptic nerve endings in which synaptic vesicles are found in close association with Rim1. B, in the model of Eliasson and co-workers, Epac2 stimulates exocytosis by interacting with secretory granule-associated SUR1 (sgSUR1), and/or pmSUR1. Both sources of SUR1 may be necessary for the cAMP-dependent regulation of ClC-3 chloride channels. Uptake of Cl⁻ into the secretory granule facilitates granule acidification and priming mediated by the v-type H⁺-ATPase.