Identification and Validation of Modulators of Exchange Protein Activated by cAMP (Epac) Activity: STRUCTURE-FUNCTION IMPLICATIONS FOR Epac ACTIVATION AND INHIBITION*

The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2′-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (R_p)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.     

Role of dynamics in the autoinhibition and activation of the exchange protein directly activated by cyclic AMP (EPAC)

The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.     

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1–4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic “C-linker,” which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V_½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or “tetrameric” C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.     

Allosteric Inhibition of Epac: COMPUTATIONAL MODELING AND EXPERIMENTAL VALIDATION TO IDENTIFY ALLOSTERIC SITES AND INHIBITORS*

Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the ability of this compound to modulate the activity of Epac and PKA, as determined by Rap1 activity and vasodilator-stimulated phosphoprotein phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that prevents the activation of Epac1 by cAMP.     

Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

Protein kinase A (PKA) holoenzyme consists of two catalytic (C) subunits and a regulatory (R) subunit dimer (R₂C₂). The kinase is activated by the binding of cAMPs to the two cyclic nucleotide binding domains (CBDs), A and B, on each R-subunit. Despite extensive study, details of the allosteric mechanisms underlying the cooperativity of holoenzyme activation remain unclear. Several Markov state models of PKA-RIα were developed to test competing theories of activation for the R₂C₂ complex. We found that CBD-B plays an essential role in R-C interaction and promotes the release of the first C-subunit prior to the binding to CBD-A. This favors a conformational selection mechanism for release of the first C-subunit of PKA. However, the release of the second C-subunit requires all four cAMP sites to be occupied. These analyses elucidate R-C heterodimer interactions in the cooperative activation of PKA and cAMP binding and represent a new mechanistic model of R₂C₂ PKA-RIα activation.     

Separate Cyclic AMP Sensors for Neuritogenesis,Growth Arrest,and Survival of Neuroendocrine Cells

Dividing neuroendocrine cells differentiate into a neuronal-like phenotype in response to ligands activating G protein-coupled receptors, leading to the elevation of the second messenger cAMP. Growth factors that act at receptor tyrosine kinases, such as nerve growth factor, also cause differentiation. We report here that two aspects of cAMP-induced differentiation, neurite extension and growth arrest, are dissociable at the level of the sensors conveying the cAMP signal in PC12 and NS-1 cells. Following cAMP elevation, neuritogenic cyclic AMP sensor/Rapgef2 is activated for signaling to ERK to mediate neuritogenesis, whereas Epac2 is activated for signaling to the MAP kinase p38 to mediate growth arrest. Neither action of cAMP requires transactivation of TrkA, the receptor for NGF. In fact, the differentiating effects of NGF do not require activation of any of the cAMP sensors protein kinase A, Epac, or neuritogenic cyclic AMP sensor/Rapgef2 but, rather, depend on ERK and p38 activation via completely independent signaling pathways. Hence, cAMP- and NGF-dependent signaling for differentiation are also completely insulated from each other. Cyclic AMP and NGF also protect NS-1 cells from serum withdrawal-induced cell death, again by two wholly separate signaling mechanisms, PKA-dependent for cAMP and PKA-independent for NGF.     

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.     

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor,Modulation by Allosteric Factors,and the Uncompetitive Inhibitor Brefeldin A

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg²⁺, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg²⁺ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg²⁺-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.     

Post-translational membrane insertion of tail-anchored transmembrane EF-hand Ca2+ sensor calneurons requires the TRC40/Asna1 protein chaperone

Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIβ activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca(2+) sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions.     

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.     

A new highly efficient substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B) reveals full autoactivation of the insulin receptor precursor

PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.     

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.     

Mapping of the localization of type 1 angiotensin receptor in membrane microdomains using bioluminescence resonance energy transfer-based sensors

Initiation and termination of signaling of the type I angiotensin receptor (AT(1)-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT(1)-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT(1)-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT(1)-R upon AngII stimulus with those of [Sar(1),Ile(8)]AngII or [Sar(1),Ile(4),Ile(8)]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT(1)-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.     

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.     

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β₁-adrenergic receptor (β₁-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β₁-AR. Recycling of the β₁-AR in AKAP5 knockdown cells was rescued by shRNA-resistant AKAP5. However, truncated mutants of AKAP5 with deletions in the domains involved in membrane targeting or in binding to calcineurin or PKA failed to restore the recycling of the β₁-AR, indicating that full-length AKAP5 was required. Furthermore, recycling of the β₁-AR in rat neonatal cardiac myocytes was dependent on targeting the AKAP5-PKA complex to the C-terminal tail of the β₁-AR. To analyze the role of AKAP5 more directly, recycling of the β₁-AR was determined in ventricular myocytes from AKAP5^−/− mice. In AKAP5^−/− myocytes, the agonist-internalized β₁-AR did not recycle, except when full-length AKAP5 was reintroduced. These data indicate that AKAP5 exerted specific and profound effects on β₁-AR recycling in mammalian cells. Biochemical or real time FRET-based imaging of cyclic AMP revealed that deletion of AKAP5 sensitized the cardiac β₁-AR signaling pathway to isoproterenol. Moreover, isoproterenol-mediated increase in contraction rate, surface area, or expression of β-myosin heavy chains was significantly greater in AKAP5^−/− myocytes than in AKAP5^+/+ myocytes. These results indicate a significant role for the AKAP5 scaffold in signaling and trafficking of the β₁-AR in cardiac myocytes and mammalian cells.     

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors

Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.     

Protein kinase A activity at the endoplasmic reticulum surface is responsible for augmentation of human ether-a-go-go-related gene product (HERG)

Human ether-a-go-go-related gene product (HERG) is a cardiac potassium channel commonly implicated in the pathogenesis of the long QT syndrome, type 2 (LQT2). LQT2 mutations typically have incomplete penetrance and affect individuals at various stages of their lives; this may mirror variations in intracellular signaling and HERG regulation. Previous work showed that sustained protein kinase A (PKA) activity augments HERG protein abundance by a mechanism that includes enhanced protein translation. To investigate the subcellular site of this regulation, we generated site-specific probes to the cytoplasmic surface of the endoplasmic reticulum (ER), the presumed locale of channel synthesis. Real-time FRET-based indicators demonstrated both cAMP and PKA activity at the ER. A PKA inhibitor targeted to the ER surface (termed p4PKIg) completely abolished PKA-mediated augmentation of HERG in HEK293 cells as well as rat neonatal cardiomyocytes. Immunofluorescence co-localization, targeted FRET-based PKA biosensors, phospho-specific antibodies, and in vivo phosphorylation experiments confirmed that p4PKIg is preferentially active at the ER surface rather than the plasma membrane. Rerouting this inhibitor to the outer mitochondrial membrane diminishes its ability to block cAMP-dependent HERG induction. Our results support a model where PKA-dependent regulation of HERG synthesis occurs at the ER surface. Furthermore, reagents generated for this study provide novel experimental tools to probe compartmentalized cAMP/PKA signaling within cells.