InteractiveFly: GeneBrief

Exchange protein directly activated by cAMP: Biological Overview | References


Gene name - Exchange protein directly activated by cAMP

Synonyms -

Cytological map position - 42D1-42D1

Function - signaling

Keywords - guanine nucleotide exchange factor - noncanonical cAMP sensor involved aversive odor learning - required for activity of edema factor (EF), an adenylate cyclase from Bacillus anthracis that blocks endocytic recycling mediated by the small GTPase Rab11 - stimulated fluid secretion in Malphigian tubules

Symbol - Epac

FlyBase ID: FBgn0085421

Genetic map position - chr2R:6,759,715-6,798,941

NCBI classification - Guanine nucleotide exchange factor for Ras-like GTPases; Cyclic nucleotide-binding domain

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

This study defines a role of the cAMP intermediate EPAC in Drosophila aversive odor learning by means of null epac mutants. Complementation analysis revealed that EPAC acts downstream from the rutabaga adenylyl cyclase and in parallel to protein kinase A. By means of targeted knockdown and genetic rescue, mushroom body Kenyon cells (KCs) were identified as a necessary and sufficient site of EPAC action. Mechanistic insights were provided by analyzing acquisition dynamics and using the 'performance increment' as a means to access the trial-based sequential organization of odor learning. Thereby it was shown that versatile cAMP-dependent mechanisms are engaged within a sequential order that correlate to individual trials of the training session (Richlitzki, 2017).

The cAMP signaling pathway is central to the regulation of plasticity and can mediate cellular responses via different intermediaries, i.e., PKA (protein kinase A), EPACs (exchange proteins directly activated by cAMP), and CNGs (cyclic nucleotide gated channels). While the numerous contributions of PKA to the regulation of plasticity have been described in great detail, the role of EPAC was not recognized until 1998 (de Rooij, 1998; Kawasaki, 1998). Since then, its operation as a noncanonical cAMP sensor has been proven in numerous studies, aided by the development of selective cAMP analogs and/or genetic models that allow discrimination between PKA and EPAC functions. Epac has been shown to enhance neurotransmitter release, activate neuronal excitability via Ca2+-dependent K+-channels, and enhance hippocampal long-term potentiation and memory consolidation. This study investigated a potential role of Epac in the Drosophila aversive odor-learning paradigm (Richlitzki, 2017).

This study has distinguish epac-dependent from epac-independent learning by means of a Drosophila null-epac mutant and used the performance increment as a means to address the functional disparity of individual training trials. Disparity impacts on the learning rate suggesting an evolutionary benefit of alternative cAMP mediators as this provides a mechanism for transforming rut-derived cAMP signals into behavioral output following different strategies (Richlitzki, 2017).

Rutabaga adenylyl cyclase (rut-AC1) is supposed to act as coincidence detector between US- and CS-derived impulses that converge at the level of KC synapses and consequently induce cAMP-dependent plasticity within an odor specific matrix of KCs. This KC synapse matrix is widely accepted as a neuronal representation of learning and thought of as an engram of an odor memory. Dopaminergic neurons (DANs) provide the major share of KCs' dopamine-induced cAMP signals, however KC synapses are not uniform but exhibit different sensitivities for cAMP. Likewise, learning is not an instantaneous process but develops over the multiple trials of a training session. As a consequence, cAMP gain over the time course of training would not be uniform but effectively determined by the sensitivity of a particular KC synapse within the odor specific matrix. This disparity would proceed to the next levels, i.e., activation gain of alternative downstream cAMP intermediaries that, in turn, are unequally sensitive as reflected by characteristic half-maximal cAMP concentrations (IC50), i.e., IC50 values for PKA ~80 nm; for HCN channels ~ 100 nm; and for EPAC ~ 800 nm (Rocher 2009; Chen, 2013). By this design, KCs diversify into high and low cAMP gain fractions that activate mediators responsive to either high and/or low cAMP amplitudes at quite different rates. Thereby, a particular odor-specific KC matrix can interconnect to multiple outputs via different mechanisms of cAMP-dependent plasticity as its synaptic elements exhibit disparate hysteresis, i.e., different time-dependent changes in the cellular cAMP levels (Richlitzki, 2017).

The surprising result that insensitive EPAC recruits early, i.e., during the second trial of the training session, suggests existence of a fast, high-amplitude cAMP signal that meets EPAC's thresholds early during training. Similarly, one has to assume that the efficacy of later training trials is mediated by a perpetuating low-amplitude signal that develops with low gain over multiple trials. Given the fact that PKA acts isogenic to EPAC and null epac mutants show regular learning during late training trials-that is from the third trial onward-neither of these intermediates is likely to account for late trial learning. One plausible mechanism would be HCN channels, i.e., subthreshold, voltage-gated ion channels that reduce membrane resistance and promote neuronal firing probability. Within sparsely firing KCs such channels might stabilize an odor-specific synaptic matrix, i.e., the CS-representation, over the 1-min time course of the training cycle and promote its transfer to mushroom body output neurons (MBONs), the recognized convergence site for KCs. In contrast, epac has been shown to enhance neurotransmitter release (Sakaba, 2003; Zhong, 2005) and/or activated neuronal excitability (Ster, 2007) via different mechanisms (Richlitzki, 2017).

While epac-dependent and independent learning mechanisms clearly dissociate at the molecular level, their learning rates appear counter-intuitive as a high-amplitude signal precedes a low-amplitude one, but both originate from rut-AC1. If one considers the animal's need to trade off certainty of a prediction against its computation time, this seemingly counterintuitive design appears deliberate and beneficial as it holds the possibility of combining both contrarian needs (DasGupta, 2014): First, epac-dependent learning requires a short computation time, i.e., after two trials, compared with the slow and cumbersome integration over multiple trials needed for epac-independent learning. Second, epac-dependent learning is restricted to salient conditions, i.e., high voltages that represent a serious threat to the animal's health, while this part of the animal's memory is spared with the 15 V DC US. In fact, wild-type strains trained with 15 V exhibited similar learning to epac-null mutants trained with 120 V suggesting that epac amplifies conditioned avoidance under trusted environmental circumstances (Richlitzki, 2017).

Do training trials clock recurrent computation within the learning network? How individual training trials are represented within the fly brain is unclear. However, functional studies have identified DANs as critical substrates of the US that tightly innervate KCs at the level of the MBs. In general, DANs and KCs work together with MBONs, the recognized readout routes of aversive odor memory. Moreover, their anatomy suggests that MBONs serve as critical inter-loops that reiterate the MBs' computational output to DANs, i.e., its major modulatory input: This recurrent connectivity exhibits a remarkable zonal architecture as dendrites of MBONs tile the length of KC axons in a nonoverlapping manner, where they meet with dopaminergic neurons (DANs), the other main innervation of the MB lobes. The DANs tile the MB lobes in a corresponding manner so that the dendrites of a particular MBON meet axonal projections of cognate DANs. Moreover, dendrites of DANs overlap with MBON axons within the MBON projection zones outside the MBs suggesting that MBONs modulate the activity of DANs and thereby generate recurrent loops. By this design MBON activity is dually modulated by DANs, first via a direct connection, and second via a KC detour that undergoes cAMP-dependent plasticity. However, further research will be required to understand the rules by which repetitive trials clock the computation within the DAN/KC/MBON network (Richlitzki, 2017).

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

Various bacterial toxins circumvent host defenses through overproduction of cAMP. A previous study has shown that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. This study provides further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies (Guichard, 2017).

Previous studies established that two cAMP toxins, EF from Bacillus anthracis and Ctx from Vibrio cholerae, block Rab11-mediated endocytic recycling of cargo such as signaling ligands and adhesion proteins, ultimately leading to inhibition of Notch signaling and loss of barrier integrity. However, the precise mechanisms by which cAMP overproduction interfered with Rab11-dependent trafficking remained to be explored. This study has examined how cAMP effector pathways converge on discrete nodes of the trafficking process subsequent to the GTP loading step to efficiently interrupt endocytic recycling (Guichard, 2017).

As is typical of small GTPases, Rab11 cycles between active (GTP-bound) and inactive (GDP-bound) conformations, the former permitting interaction with effector proteins to carry out downstream functions. Two types of regulators, activating GEFs and inactivating GAPs provide control for this essential cycle. In the particular case of Rab11, Crag (the Drosophila homolog of human DENND4A) is the only known Rab11-dedicated GEF. Similarly, only one Rab11-specific GAP has been identified: EVI5. Neither of these regulators contains an identified cAMP-binding domain that could provide a direct link between cAMP and upstream regulation of Rab11. Consistent with this inference, it was found that EF acted on Rab11 at a step subsequent to GTP loading. Indeed, transport of vesicles carrying the constitutively activated mutant Rab11*YFP were blocked by EF, while total endogenous levels of Rab11-GTP did not appear to be greatly altered (Guichard, 2017).

Association between Rab11 and its effectors Rip11 and Sec15 was abrogated by EF in several settings, including Drosophila salivary glands and human cells. The Rab11 effector Rip11 is an attractive candidate for mediating some of EF effects, as it contains a verified PKA phosphorylation site located in the central portion of the protein. Indeed, PKA-dependent phosphorylation of Rip11 is required for cAMP-potentiated insulin secretion in pancreatic β-cells. In addition, Ser/Thr phosphorylation is responsible for Rip11 transition from the insoluble to cytosolic fraction in intestinal CACO-2 cells. Although it was not determined whether the latter modification was specifically PKA-dependent, this study proposed a model in which phosphorylation of Rip11 is essential for cycling to a free state following interaction with Rab11 and specific membrane compartments prior to its re-associating with Rab11. The data show that the association between Rab11 and Rip11 can be disrupted by EF in Drosophila and mammalian endothelial or embryonic kidney cells. It is possible that unrelenting phosphorylation of Rip11 by PKA may cause the premature dissociation of Rab11 and its effectors, potentially leading to a failure to reach the AJs. While this PKA-dependent phosphorylation of Rip11 has been demonstrated in human pancreatic cells, it is not known whether it occurs in Drosophila. As dRip11 contains 19 candidate PKA phosphorylation sites, further investigation will be necessary to determine whether phosphorylation of one or more of these sites occurs and promotes the dissociation between dRip11 and Rab11. Intriguingly, Drosophila Sec15 also harbors several putative PKA phosphorylation sites, although such predicted sites are missing in its human counterpart. Importantly, this study found that artificial stimulation of Rap1 also causes a loss in Rab11*/Rip11 co-localization resulting in correlated but separated staining foci of these two proteins, suggesting that the later acting Epac/Rap1 pathway may feedback on this process (Guichard, 2017).

The second branch of the cAMP pathway mediated by the cAMP-regulated GEF Epac and its partner Rap1 (de Rooij, 2008) contributes significantly to the effect of EF in flies, and surprisingly appears to play the predominant role in mammalian systems examined in this study. In flies, activated Rap1 (Rap1*) causes a wing phenotype more similar to that of EF and Rab11DN than that of PKA*. It has been reported that Rap1* reduces the levels of Rab11 and prevents formation of Sec15 punctae (Guichard, 2013). The present study found that blocking expression of Epac significantly reduces the intensity of the EF phenotype. In addition, Rap1* alters the distribution of Rab11* and inhibits Rab11*/Rip11 co-localization. It is hypothesized that the final exocyst- and SNARE-dependent fusion event with the apical plasma membrane is subjected to inhibition by exuberant Rap1* activity, leading to accumulation of non-functional Rab11* just beneath the plasma membrane. Consistent with this hypothesis, Rap1 has been implicated by many studies in regulating of both cadherin and integrin-mediated cell-cell adhesion. Further indicating a functional connection between Rap1 signaling and Rab11-dependent trafficking, Rap1 and Rab11 over-expressed in human cells co-localize in a recent study. Additional experiments will be required to elucidate the molecular interactions connecting the activities of these two GTPases. The small GTPase RalA is a possible candidate for mediating the activity of Rap1, through activation of the Rap1 effector Rgl1, a positive regulator (GEF) of RalA. Because lowering the dose of Rgl1, or expressing a dominant-negative form of RalA, can suppress Rap1*-induced phenotypes in Drosophila, it has been proposed that RalA may act downstream of Rap1. Also, RalA is known to directly bind to exocyst components Sec5 and Exo84 and plays a central role in regulating exocyst-mediated processes in several settings, including the release of Von-Willebrand Factor from endothelial cells, or insulin secretion in pancreatic β-cells. In addition, a recent study identified Arf6 as a key component acting downstream of RalA, mediating its effect on exocyst-dependent delivery of raft micro-domains to the plasma membrane. Thus, RalA over-activationmay contribute to mediating the effect of cAMP toxins on exocyst inhibition downstream of Rap1, although this hypothesis needs to be tested in future experiments (Guichard, 2017).

Previous work showed that EF caused a drastic reduction in total Rab11 levels in wing epithelial cells (Guichard, 2010). This study found that this effect is also evident in HBMECs treated with ET, but is dependent on cell context, since inhibition of Rab11 function can be uncoupled from reduction in total Rab11 levels in Drosophila salivary glands. This reduction in Rab11 levels is unlikely to derive from transcriptional inhibition, as infection of HBMECs with Bacillis a Sterne did not result in any change in levels of Rab11 transcripts. Similarly, in Drosophila wings, where EF also triggers great reduction in Rab11 protein levels, mRNA transcript levels again were not greatly affected (Valentino Gantz, personal communication to Guichard, 2017). In HBMECs, where Rab11 levels are reduced by ET treatment, it was observed that total levels of cadherins were also severely reduced in ET-treated cells. Although the precise mechanism responsible for the loss of these proteins following ET treatment remains to be explored, it is worth noting that degradation of VE-cadherins has been observed following silencing of Rab11 in human endothelial cells, in which Rab11 is important for stabilizing cadherins at the AJs. Thus, it is possible that following EF intoxication, Rab11 and cadherins are routed to the lysosomal pathway and degraded, further impairing endocytic recycling and junctional integrity. Such an attractive hypothesis could explain the catastrophic loss of cadherins observed in ET-treated cells (Guichard, 2017).

Numerous studies have demonstrated the positive role of physiological induction of cAMP in junction establishment and stabilization, through stimulation of both PKA and Epac (Sugawara, 2009; Park, 2014). It may therefore seem counterintuitive that cAMP produced by EF or other toxins may exert an opposing effect and jeopardize junctional integrity. In principle, high versus low concentrations, sustained versus transient production, and perinuclear vs cortical subcellular distribution of toxin-delivered cAMP could elicit such opposite outcomes. In the particular case of Rab11-dependent trafficking, low physiological levels of cAMP may exert their positive effects by promoting the release of Rip11 from Rab11, as necessary to allow the final fusion event between recycling endosomes and the plasma membrane. In contrast, pathologically elevated cAMP concentrations may cause premature dissociation of the Rab11-Rip11 complex and permanently block that cycle. Similarly, uncontrolled stimulation of Rap1 by Epac could also have a negative impact on junctional transport: titration of critical partners, failure to return to complete the necessary GTP/GDP cycle, or negative feedback interference with other important steps, could explain the occurrence of this apparent paradox. Another molecule potentially at play during the response to cAMP is the small GTPase RhoA. RhoA can be phosphorylated by PKA, which inhibits its activation and prevents increased endothelial permeability during inflammation, the potential interplay between RhoA and the exocyst downstream of cAMP signaling in EF-intoxicated cells also merits further examination (Guichard, 2017).

The small GTPase Arf6 initiates retrieval of membrane proteins from cell junctions in a wide variety of cells types. Arf6, a member of the ADP-ribosylation factor subfamily, is located at the plasma membrane and some endosomal compartments, and is involved in endocytosis from the plasma membrane, vesicular recycling, and exocytosis. Importantly, Arf6 plays a role during sepsis to mediate acute VEGF-induced vascular permeability. Whether linchpin regulators of opposing vesicular trafficking pathways such as Arf6 and Rab11 interact had not yet been extensively explored. This study presents evidence that these trafficking systems do in fact engage in cross-inhibitory interactions. Consistent with the published role of Arf6 in promoting VE-cadherin endocytosis, the activated form of Arf6 (Arf6*) caused phenotypes similar to those of EF. These findings suggest that the activity of Arf6 negatively feeds back on vesicular transport to the plasma membrane by inhibiting Rab11 function. Previous studies showed that Arf6 physically interacts with the exocyst component Sec10, defining a possible avenue for the observed effects of Arf6 on Rab11 levels and distribution. Given the negative regulation of Rab11 by Arf6 in flies and its known role in compromising barrier function in the mammalian vasculature during sepsis, this study tested whether inhibitors of this pathway might antagonize the effects of EF. In human endothelial cells, it was indeed found that treatment with Slit2, a secreted peptide indirectly blocking Arf6 function, could reverse the effects of EF, restoring junctional integrity. Similarly, pharmacological inhibition of Arf6 by SecinH3, a compound that inhibits the ArfGEF ARNO, potently blocked EF-induced edema in a mouse footpad assay (Guichard, 2017).

An emerging lesson from the current and prior studies is that blocking multiple steps of branching pathways that converge on critical nodes in endocytic recycling may allow pathogens to weaken host protective mechanisms that rely on junctional integrity. For example, LF, the other toxic factor secreted by B.a, blocked exocyst-mediated vesicular docking downstream of Rab11 via inhibition of MAPK signaling. It will be interesting to explore how the various effects of EF and LF are integrated to achieve an efficient inhibition of junctional delivery, and if any compound identified in this study can also block some of the downstream effects of LF. Altogether, this study suggests that a broad range of barrier disruptive diseases ranging from cAMP related toxemia to inflammatory autoimmune diseases that involve positive feedback loops between immune activation and barrier disruption, could potentially be treated with compounds that inhibit Arf6 or Epac/Rap1, or by yet undiscovered compounds that may boost Rab11 activity (Guichard, 2017).

Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. This study, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS-bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. It is proposed that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision (Efetova, 2013).

This study has pioneered the use of bPAC, a photoactive adenylyl cyclase, as an optogenetic tool to distinguish between the functions of alternative cAMP effectors in the regulation of a physiological process in vivo. To validate bPAC as an in vivo tool Drosophila renal tubules were used to confirm the bPAC transgene could be stimulated with blue light to generate cAMP signals in a cell-type-specific manner. This optogenetic approach was combined with standard techniques that targeted PKA or EPAC to resolve the complex regulatory network of discrete cAMP pathways involved in the control of fluid secretion (Efetova, 2013).

Primary urine is generated within the main segment of the Malpighian tubules, where the principal cells establish an electrochemical gradient that provides the driving force for fluid secretion, by actively transporting potassium from the basolateral to the apical surface via a defined array of ion transporters. In parallel, the stellate cells control the anion shunt conductance and water flux of the tubules, via the action of tightly regulated aquaporins and chloride channels (Efetova, 2013).

As revealed by the current analysis, two distinct cAMP pathways are deployed within the principal cells to sustain fluid secretion: firstly, the basal principal cell PKA pathway, which regulates the rate of basal fluid secretion; and secondly the stimulatory principal cell EPAC pathway, which stimulates fluid secretion above basal levels in a cAMP-dependent manner. Manipulation of EPAC activity altered stimulated secretion but not basal secretion, and manipulation of PKA altered basal secretion but not stimulated secretion. In this respect, the two principal cell secretory control pathways appear to be independent of one another (Efetova, 2013).

Could these downstream pathways be controlled independently in vivo, through a single second messenger? While imposed cAMP signals feeding into each pathway could be generated by activation of the bPAC transgene with a defined light intensity, in vivo the neuropeptides DH44, related to corticotropin releasing factor (CRF), and DH31, related to calcitonin/calcitonin gene-related peptide (CGRP), both increase fluid secretion by raising cAMP in the principal cells. However, there is evidence in other insects that these two neuropeptides might have distinct downstream effects; in the related malarial mosquito Anopheles gambiae, DH31, but not DH44, acts as a natriuretic peptide by increasing basolateral Na+ conductance. Moreover, DH31 and DH44 have an additive stimulatory effect on fluid secretion, suggesting that they target different transport processes. Cellular association of specific GPCRs with either PKA or EPAC might well account for the different outputs observed from each GPCR. Another tempting possibility involves a class of soluble adenylyl cyclases (sACs) that are localized near the apical membrane and activated by cellular ionic concentrations rather than GPCRs, as seen in the mammalian kidney (Efetova, 2013).

The apical plasma membrane H+ V-ATPase is the driving force for ion transport in the principal cells, and is therefore an obvious downstream target for stimulatory or inhibitory cAMP signals. Formation of a functional V-ATPase complex requires PKA-dependent phosphorylation, which prevents the complex from disassembly. In blowfly salivary gland (another insect epithelium energized by a V-ATPase), cAMP has been shown to promote assembly of the V-ATPase complex. However, V-ATPase assembly - and thus activation - has also been reported via EPAC signaling within the rat renal collecting duct. By contrast, intracellular calcium has been shown to activate tubule H+ V-ATPase by directly activating mitochondria, and so increasing the ATP supply. In this complex field, optogenetic control of cellular cAMP levels in the principal cells will provide a valuable analytical tool to investigate such issues (Efetova, 2013).

A surprising feature of cAMP-dependent fluid secretion is the complete inhibition (below basal) observed with millimolar levels of cell-permeable 8-Br-cAMP, or at very high illumination levels in bPAC-transgenic tubules. Through targeted use of bPAC this effect was localized to the principal cells, and formally established an inhibitory principal cell cAMP signal. It is likely that these manipulations bring intracellular cAMP levels to abnormally high levels that are unlikely to be reached in vivo, where the resting intracellular cAMP concentration is typically in the range 0.1-1.5 μM; nonetheless, there is a real effect to be explained. At present, it is only possible to speculate on the underlying mechanisms, but it is likely that saturation or desensitization of some component of the signaling pathway is occurring; or that there is cross-talk to, for example calcium signaling via cyclic nucleotide gated calcium channels, which are known to play a role in tubule (Efetova, 2013).

In the stellate cells this study identified a stimulatory stellate cAMP signal that stimulates fluid secretion via PKA, with moderate illuminations of bPAC. In contrast, high illuminations return fluid secretion to the baseline level, suggesting that dual modulation, i.e., augmentation with low levels and inhibition with high levels of cAMP, is a common theme within the stellate and principal cells. However, further experiments will be required to substantiate this speculation (Efetova, 2013).

Interestingly, the stellate cells are known to be controlled by leucokinin, which acts though calcium, rather than cAMP, so no extracellular ligand for the stellate cAMP pathway is presently known. Tyramine has also been shown to act on stellate cells, but its second messenger is yet to be established (Efetova, 2013).

Selective elevation of cAMP in stellate cells shows that both PKA and EPAC can stimulate fluid secretion. However, these pathways do not act in parallel in the stellate cells; PKA must be upstream of EPAC, because RNAi knockdown of DC0 in stellate cells abolishes the ability of bPAC to stimulate fluid secretion. In contrast, EPAC is sufficient for secretion when activated in a cAMP-independent manner via the EPAC-specific agonist 8-pCPT-2'-O-Me-cAMP. Therefore, cAMP is likely to signal through PKA to EPAC. In turn, EPAC levels are likely to be rate limiting, as stellate-specific overexpression of epac enormously enhanced secretion (Efetova, 2013).

This study has established the use of photoactive adenylyl cyclases (PACs) as a potent tool for investigating organotypic physiological processes in vivo. A unique advantage of this optogenetic transgene is that it acts as a 'Trojan horse', allowing cell-type-specific control of cellular cAMP levels with temporal and spatial precision, through simple blue light illumination. It is this feature that has allowed deconstruction of the complex regulatory network of cAMP pathways involved in fluid secretion control, and to assign function within the Drosophila renal (Malpighian) tubule. This experimental approach can easily be adapted to other physiological preparations, for example the central nervous system or the cardiac system, to address similar physiological questions (Efetova, 2013).

Further improvements to bPAC could be achieved; for example it would be beneficial to further reduce the residual dark activity, which must be considered during experimental analysis. Although functional imaging of cAMP has been achieved, further development of this complementary technology would be advantageous for studying complex cellular signaling networks. Another feature of light-induced cAMP signals is that, as bPAC is cytoplasmic, the elevation of cAMP is uniform across the cell. In contrast, naturally occurring cAMP is often unevenly distributed on a sub-cellular level, and concentrated in local microdomains. In addition to the compartmentalization of cAMP, the cAMP sensors PKA and EPAC are also spatially regulated by binding to scaffolding proteins, such as A-kinase anchoring proteins (AKAPs). In future, it should be possible to localize genetically encoded PACs to specific subcellular domains, and embark on a new era of precision optogenetics (Efetova, 2013).

Juvenile hormone regulation of Drosophila Epac--a guanine nucleotide exchange factor

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to characterize the effects of juvenile hormone (JH) on Epac (Exchange Protein directly Activated by Cyclic AMP), a guanine nucleotide exchange factor for Rap1 in Drosophila S2 cells. JH treatment led to a rapid, dose-dependent increase in Epac relative expression ratio (RER) when compared to treatment with methyl linoleate (MLA) that lacks biological activity. The minimal level of hormone needed to elicit a response was 100 ng/ml. Time-course studies indicated a significant rise in the RER 1h after treatment. S2 cells were challenged with 20-hydroxyecdysone and a series of compounds similar in structure to JH to determine the specificity of the response. Methoprene and JH III displayed the greatest increases in RER. Late third instar (96 h) Drosophila were exposed to diet containing methoprene (500 ng/g diet); significantly higher RERs for Epac were observed 12h after exposure. JH had no effect on Epac RERs in the human cell line HEK-293 (Wang, 2009).

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited the ability to decipher Rap1-dependent pathways. In this study a procedure was developed to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1 (Dupuy, 2005).


Functions of Epac orthologs in other species

Motor learning requires Purkinje cell synaptic potentiation through activation of AMPA-receptor subunit GluA3

Accumulating evidence indicates that cerebellar long-term potentiation (LTP) is necessary for procedural learning. However, little is known about its underlying molecular mechanisms. Whereas AMPA receptor (AMPAR) subunit rules for synaptic plasticity have been extensively studied in relation to declarative learning, it is unclear whether these rules apply to cerebellum-dependent motor learning. This study shows that LTP at the parallel-fiber-to-Purkinje-cell synapse and adaptation of the vestibulo-ocular reflex depend not on GluA1- but on GluA3-containing AMPARs. In contrast to the classic form of LTP implicated in declarative memory formation, this form of LTP does not require GluA1-AMPAR trafficking but rather requires changes in open-channel probability of GluA3-AMPARs mediated by cAMP signaling and activation of the protein directly activated by cAMP (Epac). It is concluded that vestibulo-cerebellar motor learning is the first form of memory acquisition shown to depend on GluA3-dependent synaptic potentiation by increasing single-channel conductance (Gutierrez-Castellanos, 2017).

Epac signaling is required for cocaine-induced change in AMPA receptor subunit composition in the ventral tegmental area

Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). This study tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. A single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. This study also showed that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning (Liu, 2016).

beta-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1alpha interaction to potentiate glutamate release at cerebrocortical nerve terminals

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. This study describes the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1alpha and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the beta-adrenergic receptor (betaAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of betaARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, it is concluded that betaARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals (Ferrero, 2013).

Identification and characterization of small molecules as potent and specific EPAC2 antagonists

EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP (EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes under physiological and pathophysiological circumstances. This study report the chemical design, synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC(50) of 0.3 muM for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition of EPAC1-mediated Rap1-GDP exchange activity at 25 muM, indicating that they are EPAC2-specific antagonists. Moreover, live-cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific antagonist (Chen, 2013).

EPAC signalling pathways are involved in low PO2 chemoreception in carotid body chemoreceptor cells

Chemoreceptor cells of the carotid bodies (CB) are activated by hypoxia and acidosis, responding with an increase in their rate of neurotransmitter release, which in turn increases the electrical activity in the carotid sinus nerve and evokes a homeostatic hyperventilation. Studies in isolated chemoreceptor cells have shown that moderate hypoxias (46 mmHg) produces smaller depolarisations and comparable Ca(2+) transients but a much higher catecholamine (CA) release response in intact CBs than intense acidic/hypercapnic stimuli (20% CO(2), pH 6.6). Similarly, intense hypoxia (20 mmHg) produces smaller depolarizations and Ca(2+) transients in isolated chemoreceptor cells but a higher CA release response in intact CBs than a pure depolarizing stimulus (30-35 mm external K(+)). Studying the mechanisms responsible for these differences this study has found the following. (1) Acidic hypercapnia inhibited I(Ca) (60%; whole cell) and CA release (45%; intact CB) elicited by ionomycin and high K(+). (2) Adenylate cyclase inhibition (SQ-22536; 80 microm) inhibited the hypoxic release response (>50%) and did not affect acidic/hypercapnic release, evidencing that the high gain of hypoxia to elicit neurotransmitter release is cAMP dependent. (3) The last effect was independent of PKA activation, as three kinase inhibitors did not alter the hypoxic release response. (4) The Epac (exchange protein activated by cAMP) activator reversed the effects of the cyclase inhibitor. (5) The Epac inhibitor brefeldin A (100 microm) inhibited (54%) hypoxic induced release. These findings show for the first time that an Epac-mediated pathway mediates O(2) sensing/transduction in chemoreceptor cells (Rocher, 2009).

Exchange protein activated by cAMP (Epac) mediates cAMP activation of p38 MAPK and modulation of Ca2+-dependent K+ channels in cerebellar neurons

The exchange factor directly activated by cAMP (Epac) is a newly discovered direct target for cAMP and a guanine-nucleotide exchange factor for the small GTPase Rap. Little is known about the neuronal functions of Epac. This study shows that activation of Epac by specific cAMP analogs or by the pituitary adenylate cyclase-activating polypeptide induces a potent activation of the Ca2+-sensitive big K+ channel, slight membrane hyperpolarization, and increased after-hyperpolarization in cultured cerebellar granule cells. These effects involve activation of Rap and p38 MAPK, which mobilizes intracellular Ca2+ stores. These findings reveal a cAMP Epac-dependent and protein kinase A-independent signaling cascade that controls neuronal excitability (Ster, 2007).

cAMP acts on exchange protein activated by cAMP/cAMP-regulated guanine nucleotide exchange protein to regulate transmitter release at the crayfish neuromuscular junction

Glutamatergic synapses are highly modifiable, suiting them for key roles in processes such as learning and memory. At crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) ion channels mediate hormonal modulation of glutamatergic synapses and a form activity-dependent long-term facilitation (LTF) of synaptic transmission. This study shows that a new target for cAMP, exchange protein activated by cAMP (Epac) or cAMP-regulated guanine nucleotide exchange protein, is involved in the hormonal enhancement of synaptic transmission by serotonin. Induction of LTF 'tags' synapses, rendering them responsive to cAMP in an HCN-independent manner. Epac also mediates the enhancement of tagged synapses. Thus, the cAMP-dependent enhancement of transmission is mediated by two separate pathways, neither of which involves protein kinase A (Zhong, 2005).

Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP

Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. This study reports that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). A gene encoding a guanine-nucleotide-exchange factor (GEF) has been named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. It is concluded that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP (de Rooij, 1998).


REFERENCES

Search PubMed for articles about Drosophila Epac

Chen, H., Tsalkova, T., Chepurny, O. G., Mei, F. C., Holz, G. G., Cheng, X. and Zhou, J. (2013). Identification and characterization of small molecules as potent and specific EPAC2 antagonists. J Med Chem 56(3): 952-962. PubMed ID: 23286832

DasGupta, S., Ferreira, C. H. and Miesenbock, G. (2014). FoxP influences the speed and accuracy of a perceptual decision in Drosophila. Science 344(6186): 901-904. PubMed ID: 24855268

de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A. and Bos, J. L. (1998). Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396(6710): 474-477. PubMed ID: 9853756

Dupuy, A. G., L'Hoste, S., Cherfils, J., Camonis, J., Gaudriault, G. and de Gunzburg, J. (2005). Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac. Oncogene 24: 4509-4520. PubMed ID: 15856025

Efetova, M., Petereit, L., Rosiewicz, K., Overend, G., Haussig, F., Hovemann, B. T., Cabrero, P., Dow, J. A. and Schwarzel, M. (2013). Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo. J Cell Sci 126(Pt 3): 778-788. PubMed ID: 23264735

Ferrero, J. J., Alvarez, A. M., Ramirez-Franco, J., Godino, M. C., Bartolome-Martin, D., Aguado, C., Torres, M., Lujan, R., Ciruela, F. and Sanchez-Prieto, J. (2013). beta-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1alpha interaction to potentiate glutamate release at cerebrocortical nerve terminals. J Biol Chem 288(43): 31370-31385. PubMed ID: 24036110

Guichard, A., McGillivray, S. M., Cruz-Moreno, B., van Sorge, N. M., Nizet, V. and Bier, E. (2010). Anthrax toxins cooperatively inhibit endocytic recycling by the Rab11/Sec15 exocyst. Nature 467(7317): 854-858. PubMed ID: 20944747

Guichard, A., Jain, P., Moayeri, M., Schwartz, R., Chin, S., Zhu, L., Cruz-Moreno, B., Liu, J. Z., Aguilar, B., Hollands, A., Leppla, S. H., Nizet, V. and Bier, E. (2017). Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport. PLoS Pathog 13(9): e1006603. PubMed ID: 28945820

Gutierrez-Castellanos, N., Da Silva-Matos, C. M., Zhou, K., Canto, C. B., Renner, M. C., Koene, L. M. C., Ozyildirim, O., Sprengel, R., Kessels, H. W. and De Zeeuw, C. I. (2017). Motor learning requires Purkinje cell synaptic potentiation through activation of AMPA-receptor subunit GluA3. Neuron 93(2): 409-424. PubMed ID: 28103481

Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E. and Graybiel, A. M. (1998). A family of cAMP-binding proteins that directly activate Rap1. Science 282(5397): 2275-2279. PubMed ID: 9856955

Liu, X., Chen, Y., Tong, J., Reynolds, A. M., Proudfoot, S. C., Qi, J., Penzes, P., Lu, Y. and Liu, Q. S. (2016). Epac signaling is required for cocaine-induced change in AMPA receptor subunit composition in the ventral tegmental area. J Neurosci 36(17): 4802-4815. PubMed ID: 27122037

Park, S. W., Schonhoff, C. M., Webster, C. R. and Anwer, M. S. (2014). Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane. Am J Physiol Gastrointest Liver Physiol 307(8): G863-870. PubMed ID: 25190474

Richlitzki, A., Latour, P. and Schwarzel, M. (2017). Null EPAC mutants reveal a sequential order of versatile cAMP effects during Drosophila aversive odor learning. Learn Mem 24(5): 210-215. PubMed ID: 28416632

Rocher, A., Caceres, A. I., Almaraz, L. and Gonzalez, C. (2009). EPAC signalling pathways are involved in low PO2 chemoreception in carotid body chemoreceptor cells. J Physiol 587(Pt 16): 4015-4027. PubMed ID: 19581380

Sakaba, T. and Neher, E. (2003). Direct modulation of synaptic vesicle priming by GABA(B) receptor activation at a glutamatergic synapse. Nature 424(6950): 775-778. PubMed ID: 12917685

Ster, J., De Bock, F., Guerineau, N. C., Janossy, A., Barrere-Lemaire, S., Bos, J. L., Bockaert, J. and Fagni, L. (2007). Exchange protein activated by cAMP (Epac) mediates cAMP activation of p38 MAPK and modulation of Ca2+-dependent K+ channels in cerebellar neurons. Proc Natl Acad Sci U S A 104(7): 2519-2524. PubMed ID: 17284589

Sugawara, K., Shibasaki, T., Mizoguchi, A., Saito, T. and Seino, S. (2009). Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis. Genes Cells 14(4): 445-456. PubMed ID: 19335615

Wang, J., Lindholm, J. R., Willis, D. K., Orth, A. and Goodman, W. G. (2009). Juvenile hormone regulation of Drosophila Epac--a guanine nucleotide exchange factor. Mol Cell Endocrinol 305(1-2): 30-37. PubMed ID: 19433259

Zhong, N. and Zucker, R. S. (2005). cAMP acts on exchange protein activated by cAMP/cAMP-regulated guanine nucleotide exchange protein to regulate transmitter release at the crayfish neuromuscular junction. J Neurosci 25(1): 208-214. PubMed ID: 15634783


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date revised: 26 September 2018

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