Macromolecular Signaling Complexes Research Articles

EPAC1 controls vascular endothelial cell permeability and the adaptation of these cells to differential fluid shear stress

The permeability of a monolayer of vascular endothelial cells (VECs) to the paracellular transit of macromolecules is largely dependent on their ability to form vascular endothelial cadherin (VEcad)‐based contacts between neighbouring VECs and on the strength of the linkages between these VECad‐based structures and the actin cytoskeleton. We reported previously that cAMP‐elevating agents reduce VEC permeability largely through an exchange protein activated by cAMP‐1 (EPAC1)‐mediated strengthening of these inter‐VEC adhesions. Specifically, we showed that integration of EPAC1 into VECad‐based macromolecular signaling complexes allowed localized EPAC1‐dependent activation of Rap‐1 at regions of VEC intercellular contacts and reduced permeability. Since high VEC permeability occur in vivo in areas of the vasculature that experience low fluid shear‐stresses and these areas are more likely to develop atherosclerotic lesions, we studied the impact of EPAC1 activation on VEC permeability under conditions of high or low laminar shear stress. Our results indicated that while VECs aligned poorly when subjected to low fluid shear‐stresses, EPAC1 activators markedly promoted alignment under these conditions.

The Adaptor MAVS Promotes NLRP3 Mitochondrial Localization and Inflammasome Activation

NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1β. The adaptor ASC is necessary for NLRP3-dependent inflammasome function, but it is not known whether ASC is a sufficient partner and whether inflammasome formation occurs in the cytosol or in association with mitochondria is controversial. Here, we show that the mitochondria-associated adaptor molecule, MAVS, is required for optimal NLRP3 inflammasome activity. MAVS mediates recruitment of NLRP3 to mitochondria, promoting production of IL-1β and the pathophysiologic activity of the NLRP3 inflammasome in vivo. Our data support a more complex model of NLRP3 inflammasome activation than previously appreciated, with at least two adapters required for maximal function. Because MAVS is a mitochondria-associated molecule previously considered to be uniquely involved in type 1 interferon production, these findings also reveal unexpected polygamous involvement of PYD/CARD-domain-containing adapters in innate immune signaling events.

Probing G Protein-Coupled Receptor Signalling Complex Dynamics and Cellular Outcomes in Living Cells using Bioluminescence Resonance Energy Transfer (BRET)

G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signals transduction across biological membranes. In recent years, it has become clear that GPCRs are not uni-dimensional switches that turn ‘on’ or ‘off’ a single signalling pathway. Instead, each receptor can engage multiple signalling partners to form dynamic signalling complexes that can engage various downstream effector systems. Individual ligands can have differential efficacies toward specific subsets of the signalling effectors that can be regulated by a given receptor. This phenomenon, known as ligand-biased signalling, opens new opportunities for the development of new drugs with increased selectivity profiles and less undesirable effects. Such functional selectivity is in part based on the formation of distinct signalling complexes promoted by various ligands. In an effort to better understand the structural and molecular basis of GPCR functional selectivity, we developed a diversity of biosensors based on bioluminescence resonance energy transfer (BRET) that allow real-time monitoring of the interactions between GPCRs and multiple effectors as well as the downstream signalling events. The biosensors can be divided in uni- and multi-molecular units that can sense conformational rearrangements resulting from the binding of a signalling partner, a second messenger or from the occurrence of a post-translational modification. They are also well suited to monitor the protein-protein interactions occurring within macromolecular signalling complexes. These biosensors can directly monitor the activation of a large diversity of signalling pathways in real time. To date we generated more than 30 BRET-based sensors that monitor various aspects of dynamic signalling complexes assembly and their resulting cellular effects. Their use revealed unexpected new signalling complexes and provide a new tool set to monitor cellular signalling from the membrane to the nucleus.

Abstract 19025: A Signaling Complex of HCN2/HCN4/Cav3 Resides in Caveolae of Cardiac Cells

Macromolecular signaling complexes including ion channels present in the caveolar micro-domains can provide highly specific and localized regulation of cell function. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key constituents of the cardiac pacemaker, regulating the rate of diastolic depolarization and cardiac rhythm. HCN4 and HCN2 are the major HCN isoforms found in cardiac tissues. HCN4 is physically associated with Caveolin-3 (Cav3), the “coat protein” for caveolae in cardiac cells. In this report, we dissected the regulation of Cav3 on HCN2. Cardiac HCN2 (60-kDa, HCN2s) or HCN4 was co-localized with Cav3 in mouse LV membrane fractions. A dominant-negative P104L-Cav3 mutation was used to “probe” the association of HCN2/Cav3. HCN2s itself does not encode currents but HCN2s/HCN4 channels generated currents resembled to those recorded from native cells. HEK293 cells expressing full-length-HCN2 (HCN2f)/WTCav3 showed comparable HCN2 density and channel activity to those cells expressing HCN2f/P104L or HCN2 alone. Bioluminescence Resonance Energy Transfer (BRET) assays in conjunction with stable HCN cell lines and lipid raft inhibitors were used to quantify the intra-molecular interactions of each HCN form and Cav3. WTCav3 or P104L was fused to a Renilla Luciferase (Rluc) as the “donor” while each HCN form was fused to YFP as the “acceptor”. All HCN/Cav3 pairs displayed positive BRET ratios, suggesting that HCN and Cav3 were within a 10-100 Å distance to permit energy transfer from RLuc (470-nm) to YFP (530-nm) in the presence of coelenterazine-h. We did not record positive BRET ratios from samples containing no assay substrate. Average BRET ratio data (n=6, p<0.05) suggested that the association for HCN2s/Cav3 is the strongest. The P104L study showed that aa104 is involved in the interactions of HCN2s/Cav3 or HCN4/Cav3 but not HCN2f/Cav3. Our results indicate that Cav3 interacts with HCN4 via HCN2s to form a HCN2s/HCN4/Cav3/caveolae complex, and it may play an important role in cardiac signaling.

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

Virtual Screening for LPA2-Specific Agonists Identifies a Nonlipid Compound with Antiapoptotic Actions

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA₂ G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA₂ receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA(1&2) double-knockout mice reconstituted with the LPA₂ receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA₂, Na⁺ - H⁺ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA₂-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

<em>In Vitro</em> Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis(1-3). CFTR has been implicated in two major diseases: cystic fibrosis (CF)(4) and secretory diarrhea(5). In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States(6). Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut(7). Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo(8-19). In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP(20-27). The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)(20). Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities(22). This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells(16-18). Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay(16-19,28,29). Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR(16-19,28,29).

Translocator protein (18 kDa), a potential molecular imaging biomarker for non-invasively distinguishing non-alcoholic fatty liver disease

Mitochondrial dysfunction is responsible for liver damage and disease progression in non-alcoholic fatty liver disease (NAFLD). Translocator protein (18 kDa) (TSPO), a mitochondrial transmembrane protein, plays important roles in modulating mitochondrial function. This study explored whether TSPO can be used as an imaging biomarker of non-invasive diagnosis and staging of NAFLD, monitored using positron emission tomography (PET) with a TSPO radioligand [(18)F]FEDAC. PET with [(18)F]FEDAC, non-enhanced computerized tomography (CT), autoradiography, histopathology, and gene analysis were performed to evaluate and quantify TSPO levels and NAFLD progression in methionine and choline-deficient diet-fed mice. Correlations were analyzed between uptake ratio of radioactivity and NAFLD activity score (NAS) in the liver. Uptake of [(18)F]FEDAC obviously increased with disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) (p<0.01). A close correlation was identified between [(18)F]FEDAC uptake ratio and NAS in the liver (Pearson's r=0.922, p=0.000). Specific binding of [(18)F]FEDAC to TSPO in the NAFLD livers was assessed in competition studies with the unlabelled TSPO-selective ligand PK11195. Autoradiography and histopathology confirmed the PET imaging results. Further, the mRNA levels of the functional macromolecular signaling complex composed of TSPO were obviously higher compared to controls. TSPO expression increases in NAFLD and closely correlates with NAFLD progression. TSPO as a specific molecular imaging biomarker may open a novel avenue for non-invasive, reliable, and quantitative diagnosis and staging of NAFLD.

Cyclic Nucleotide Phosphodiesterase 3 Signaling Complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.

Bivalent binding drives the formation of the Grb2–Gab1 signaling complex in a noncooperative manner

Although the growth factor receptor binder 2 (Grb2)-Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer-dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be structurally disordered, it nonetheless adopts a more or less compact conformation reminiscent of natively folded globular proteins. Importantly, the structurally flexible conformation of the PR domain appears to facilitate the binding of Gab1 to Grb2 with a 1:2 stoichiometry. More specifically, the formation of the Grb2-Gab1 signaling complex is driven via a bivalent interaction through the binding of the C-terminal homology 3 (cSH3) domain within each monomer of Grb2 homodimer to two distinct RXXK motifs, herein designated G1 and G2, located within the PR domain of Gab1. Strikingly, in spite of the key role of bivalency in driving this macromolecular assembly, the cSH3 domains bind to the G1 and G2 motifs in an independent manner with zero cooperativity. Taken together, our findings shed new light on the physicochemical forces driving the assembly of a key macromolecular signaling complex that is relevant to cellular health and disease.

Mechanisms of Radiomitigative Cell Signaling Via Lysophosphatidic Acid Receptors

Radiomitigators attenuate radiation injury when administered after irradiation. Lysophosphatidic acid (LPA) a lipid mediator rescues apoptotically condemned cells. LPA protects IEC‐6 intestinal epithelial cells in vitro and intestinal crypts in vivo from radiation‐induced apoptosis. Using KO mice and cells we established that the LPA2 receptor is required for radiomitigation. Through its C‐terminal PDZ and LIM‐binding motifs LPA2 forms agonist‐dependent macromolecular signaling complexes. LPA2 interacts with the proapoptotic protein Siva‐1 and targets it for proteasomal degradation. Full activation of the NFkB and ERK1/2 prosurvival pathway requires formation of an LPA2, NHERF2, and TRIP‐6 ternary macromolecular complex. These signals inhibit the mitochondrial apoptosis pathway. Reconstitution of the LPA2 receptor into MEFs derived from LPA1/2 KO mice restores radioprotection. Conditioned medium from irradiated U937 cells induces bystander apoptosis in non‐irradiated IEC‐6 cultures and LPA2‐transfected MEFs. Using in silico drug discovery we found a novel LPA2‐selective agonist GRI977143 that protects cells from radiation‐ and chemotherapy‐induced apoptosis. GRI977143 also increases the survival of mice exposed to lethal levels of radiation. Supported by AI08405.

Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95

N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.

Multivalent Binding and Facilitated Diffusion Account for the Formation of the Grb2–Sos1 Signaling Complex in a Cooperative Manner

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein-protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein-protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein-protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein-protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein-protein interactions in a dynamic context- and phosphorylation site-specific manner.

Using Multi-Color Super-Resolution Microscopy to Probe the Organization of Dyadic Proteins within Rat Cardiac Myocytes

Cardiac excitation-contraction coupling relies on calcium release from internal stores (the sarcoplasmic reticulum -SR) via ryanodine receptors (RyRs), which are intracellular calcium channels. Most RyRs are organized into clusters within narrow dyadic junctions between sarcolemmal and sarcoplasmic reticulum (SR) membranes. It is still unclear how junctions are formed and maintained. One protein that has been implicated in dyadic junction formation is the cardiac isoform of the junctophilin family, junctophilin-2 (JPH2). With the increased resolution available in multi-color super-resolution microscopy it is now possible to investigate the nanoscale relationship between proteins such as RyRs and JPH2 which are found within the junction. Using single fluorophore localization based super-resolution microscopy we have studied the distribution of a number of such proteins in rat ventricular myocytes.Analysing the peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), we have found that JPH2 was almost completely associated with RyR clusters. Estimates of co-localization of ∼89% between JPH2 and RyRs confirmed near complete colocalization within peripheral couplons. The shape of associated RyR clusters and JPH2 clusters were very similar, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked. Our data shows, with nanoscale resolution, that junctophilin is not constrained to specific anchoring domains within the junction, but is interspersed throughout the entire area occupied by RyRs.These results demonstrate the utility of fluorescent multi-colour super-resolution immuno-labeling to investigate protein proximity at the nanometer scale. The ability to resolve the association of different molecules into the same biological structure at the nanoscale (here the dyad junction) should give new insight into the assembly and function of macromolecular signaling complexes.

Abstract 8556: Inhibition of Grk2 Augments Cardiac Beta2-Adrenergic Receptor-Dependent Contractility via Prevention of Receptor Interaction with Phosphodiesterase Type 4d

β 1 - and β 2 -adrenergic receptors (βARs) are G-protein coupled receptors (GPCRs) that play clearly distinct roles in cardiac physiology/pathology. For instance, cardiomyocyte contraction is readily stimulated by β 1 AR but not β 2 AR signaling. This might be explained by differences in assembly of macromolecular signaling complexes: β 1 AR forms a signaling complex with phosphodiesterase (PDE) type 4D8 directly, and agonist binding dissociates this complex. Conversely, GPCR kinase (GRK)2-induced β 2 AR phosphorylation leads to recruitment of a complex consisting of β-arrestin (βarr), a universal GPCR adapter/scaffolding molecule, and another PDE4D variant, PDE4D5. This PDE4D recruitment is postulated to constrain β 2 AR pro-contractile signaling by limiting compartmentalization of 3′-5′-adenosine monophosphate signaling. Thus, herein, we sought to investigate the effect of inhibition of this complex recruitment on β 2 AR pro-contractile signaling in vivo. In order to block β 2 AR-PDE4D interaction in cardiac myocytes in vivo, we crossed β 1 AR knockout (B1KO) mice, which do not express β 1 AR, with M27 mice, which overexpress, specifically in cardiac myocytes, the GRK2 inhibitor GRK2ct (or βARKct). By blocking GRK2-mediated phosphorylation, βarr-dependent PDE4D recruitment to β 2 AR is prevented. We studied the offspring both under normal conditions and after surgically induced myocardial infarction (MI). Contractility was significantly augmented in M27/B1KO mice compared to B1KO's, both in healthy mice (ejection fraction (EF): 69+1.8% vs. 57+1.7% respectively, p&lt;0.05, n=8) and at 4 weeks after MI (EF: 42.6+0.1% vs. 25+3.9% respectively, p&lt;0.05, n=8). In addition, GRK2ct expression attenuated cardiac dilatation of B1KO's at 4 weeks post-MI. At the molecular level, M27/B1KO hearts displayed significantly less membrane recruitment of both PDE4D5 and PDE4D3 compared to B1KO hearts, as evidenced by western blotting in cardiac membrane preparations, indicating less β 2 AR-PDE4D interaction. Thus, cardiac GRK2 inhibition by GRK2ct increases β 2 AR-dependent contractility both normally and in post-MI heart failure. The underlying mechanism appears to involve inhibition of the GRK2/βarr-mediated PDE4D recruitment to the receptor.

Abstract 16943: Dissecting the Molecular Interaction of Caveolin3 and the Regulatory Subunit (SUR2) of Cardiac KATP Channels

Macromolecular signaling complexes including ion channels present in caveolar microdomains can provide highly specific and localized regulation of cell function. Prior studies have shown that Caveolin3 (Cav3) may interact with a Kir6.2 pore of the cardiac ATP-sensitive potassium channels (KATP). In this report, we studied the molecular interaction of Cav3 and SUR2, the regulatory subunit of KATP. Cav3 and SUR2 were detected in the same caveolar fractions isolated from mouse left ventricles. A P104L-Cav3 mutation was used to probe Cav3-SUR2 trafficking and molecular interaction in COS1 cells. Immunostaining data showed that SUR2 co-localized with Cav3, however, P104L caused retention of SUR2-specific fluorescent signals intracellularly. A quantitative Bioluminescence Resonance Energy Transfer (BRET) assay using coelenterazine-h as substrate was established to study Cav3-SUR2 interaction. WT or P104L-Cav3 was fused to a Renilla Luciferase (Rluc) while SUR2 was fused to YFP. Both WTCav3/SUR2 (0.19±0.04, n=10) and P104LCav3/SUR2 (0.17±0.02, n=10, P=0.05) pairs displayed positive BRET ratios, suggesting that the two proteins were within a 10-100 Å distance to permit energy transfer from RLuc (470-nm) to YFP (530-nm). We did not record positive BRET ratios from negative control samples containing no substrate. Our data showed that Cav3 could directly bind to SUR2, and this interaction was not significantly affected by the P104L mutation, indicating that aa104 is probably not a binding site for the two proteins. To determine which portion of SUR2 binds to Cav3, 3 expression clones harboring SUR2 transmembrane domain TMD0, TMD1 or TMD2 were made. BRET results showed that Cav3 preferably binds to TMD0 (0.19±0.01, n=5) or TMD1 (0.15±0.03, n=5) over TMD2 (0.02±0.01, n=5). Co-immunoprecipitation results confirmed our BRET data. Mouse neonatal myocytes infected with adenoviral particles expressing P104LCav3 displayed a significantly prolonged action potential (APD90, 20.5±4, n=6) than WTCav3-expressing cells (9.3±4, n=4, p&lt;0.05), which suggested that Cav3 levels affected membrane electrical properties. Together with other data, a Cav3-SUR2-Kir6.2 complex that resides in the caveolar microdomains may play an important role in cardiac signaling.

The Structure of the PDZ3-SH3-GuK Tandem of ZO-1 Protein Suggests a Supramodular Organization of the Membrane-associated Guanylate Kinase (MAGUK) Family Scaffold Protein Core

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tethering membrane receptors, adhesion molecules, and macromolecular signaling complexes for tissue developments, cell-cell communications, and intracellular signal transductions. The defining feature of the MAGUK family scaffolds is that each member contains a conserved core consisting of a PSD-95/Dlg/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase (GuK) domain arranged in tandem, although the structural features and functional implications of the PDZ-SH3-GuK tandem arrangement are unclear. The structure of the ZO-1 PDZ3-SH3-GuK tandem solved in this study reveals that the PDZ domain directly interacts with the SH3-GuK module, forming a structural supramodule with distinct target binding properties with respect to the isolated domains. Structure-based sequence analysis suggests that the PDZ-SH3-GuK tandems of other members of the MAGUK family also form supramodules.

A-kinase Anchoring Proteins: Temporal and Spatial Regulation of Intracellular Signal Transduction in the Cardiovascular System

The prototypical intracellular second messenger cAMP is locally controlled by multimolecular protein complexes organized by A-kinase anchoring proteins (AKAPs). AKAPs serve as scaffolds for different sets of cAMP-metabolizing enzymes that control cAMP gradients and regulate cellular responses. In addition to adenylyl cyclases and phosphodiesterases, AKAPs bind signaling enzymes and ion channels that are important regulators of cardiac contractility and pathophysiological myocyte remodeling and hypertrophy. Compartmentation increases the local concentration of cAMP signaling components, providing faster, higher fidelity regulation of cellular processes. This review series highlights the contribution of AKAPs in the heart as scaffold proteins integrating protein kinases, phosphatases and other effector molecules involved in cAMP-dependent signaling.