Key G Protein-coupled Receptors Research Articles

Structural dynamics of chemokine receptors.

Membrane proteins such as G protein-coupled receptors (GPCRs) are involved in awide range of physiological and pathological cellular processes. Binding of extracellular signals to GPCRs, including hormones, neurotransmitters, peptides and proteins, can activate intracellular signaling cascades via G protein interaction. Chemokine receptors are key GPCRs implicated in cancers, immune responses, cell migration and inflammation. Specifically, the CCR5 and CXCR4 chemokine receptors serve as important therapeutic targets against Human Immunodeficiency virus (HIV) entry into human cells. Maraviroc and Vicriviroc, two clinically used HIV entry inhibitors, are antagonists of the CCR5 receptor. These drugs block HIV entry, but ultimately resistance develops, due to emergence of viruses that can utilize the CXCR4 co-receptor. Unfortunately, development of chemokine receptor antagonists as selective drugs of HIV infection has been greatly hindered as their target orthosteric site is conserved among different receptor subtypes. Accordingly, it is important to understand the structural dynamics of these receptors to develop more effective therapeutics. In this chapter, we describe the latest advances in studies of these two key chemokine receptors with respect to their structures, dynamics and function.

Multi-Omics Analysis for Transcriptional Regulation of Immune-Related Targets Using Epigenetic Data: A New Research Direction.

BackgroundCurrently, a comprehensive method for exploration of transcriptional regulation has not been well established. We explored a novel pipeline to analyze transcriptional regulation using co-analysis of RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq).MethodsThe G protein-coupled receptors (GPCRs) possibly associated with macrophages were further filtered using a reduced-Cox regression model. ATAC-seq profiles were used to map the chromatin accessibility of the GPRC5B promoter region. Pearson analysis was performed to identify the transcription factor (TF) whose expression was correlated with open chromatin regions of GPRC5B promoter. ChIP-seq profiles were obtained to confirm the physical binding of GATA4 and its predicted binding regions. For verification, quantitative polymerase chain reaction (qPCR) and multidimensional database validations were performed.ResultsThe reduced-Cox regression model revealed the prognostic value of GPRC5B. A novel pipeline for TF exploration was proposed. With our novel pipeline, we first identified chr16:19884686-19885185 as a reproducible open chromatin region in the GPRC5B promoter. Thereafter, we confirmed the correlation between GATA4 expression and the accessibility of this region, confirmed its physical binding, and proved in vitro how its overexpression could regulate GPRC5B. GPRC5B was significantly downregulated in colon adenocarcinoma (COAD) as seen in 28 patient samples. The correlation between GPRC5B and macrophages in COAD was validated using multiple databases.ConclusionGPRC5B, correlated with macrophages, was a key GPCR affecting COAD prognosis. Further, with our novel pipeline, TF GATA4 was identified as a direct upstream of GPRC5B. This study proposed a novel pipeline for TF exploration and provided a theoretical basis for COAD therapy.

Retrospective ensemble docking of allosteric modulators in an adenosine G-protein-coupled receptor

BackgroundEnsemble docking has proven useful in drug discovery and development. It increases the hit rate by incorporating receptor flexibility into molecular docking as demonstrated on important drug targets including G-protein-coupled receptors (GPCRs). Adenosine A1 receptor (A1AR) is a key GPCR that has been targeted for treating cardiac ischemia-reperfusion injuries, neuropathic pain and renal diseases. Development of allosteric modulators, compounds binding to distinct and less conserved GPCR target sites compared with agonists and antagonists, has attracted increasing interest for designing selective drugs of the A1AR. Despite significant advances, more effective approaches are needed to discover potent and selective allosteric modulators of the A1AR. MethodsEnsemble docking that integrates Gaussian accelerated molecular dynamic (GaMD) simulations and molecular docking using Autodock has been implemented for retrospective docking of known positive allosteric modulators (PAMs) in the A1AR. ResultsEnsemble docking outperforms docking of the receptor cryo-EM structure. The calculated docking enrichment factors (EFs) and the area under the receiver operating characteristic curves (AUC) are significantly increased. ConclusionsReceptor ensembles generated from GaMD simulations are able to increase the success rate of discovering PAMs of A1AR. It is important to account for receptor flexibility through GaMD simulations and flexible docking. General significanceEnsemble docking is a promising approach for drug discovery targeting flexible receptors.

Phospho‐proteomic Analysis of Protease‐activated Receptor‐1 Biased Signaling Reveals Novel Modulators of Endothelial Barrier Function

Dysregulation of the vascular endothelium is a hallmark of inflammation and promotes barrier disruption, vascular leakage and tissue edema. Vascular endothelial dysfunction is mediated by vasoactive agents and inflammatory mediators, many of which signal through G protein‐coupled receptors (GPCRs). A key GPCR inflammatory mediator is thrombin, a procoagulant protease generated in response to vascular injury and inflammation. Thrombin activates protease‐activated receptor‐1 (PAR1) in endothelial cells to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC) is an anticoagulant protease that activates PAR1 through a distinct mechanism and promotes endothelial barrier stabilization. The ability of distinct tethered ligands formed through cleavage of PAR1 by different proteases results in stabilization of unique receptor active conformational states that promote opposite signaling effects is termed biased signaling. Despite progress in understanding PAR1 bias signaling in endothelial cells, the proteins and pathways by which thrombin and APC regulate the endothelial barrier integrity are largely unknown. Here, we report the global phosphoproteome of thrombin and APC signaling using tandem‐mass‐tag (TMT) mass spectrometry and computational analyses. Our results reveal unique temporal dynamics profiles of thrombin and APC signaling and the identification of multiple enriched phosphoproteome associated biological functions including microtubules, adherens junctions, actin and signaling, all key modulators of endothelial barrier permeability. We selected candidates that showed statistically significant changes in phosphorylation following thrombin or APC stimulation, and validated the function of four proteins including: afadin (AFDN), CDC42 binding protein kinase alpha (BPA), adducin 1 (ADD1), and GPCR interacting protein 1 (GIT1) using an siRNA‐targeted approach. Using target‐specific siRNAs to deplete candidate proteins in endothelial cells, we found that depletion of afadin and adducin had statistically significant effects on endothelial barrier permeability. Afadin depletion led to an increase in thrombin‐induced permeability, while adducin depletion virtually ablated thrombin‐stimulated permeability. Immunofluorescence microscopy imaging of morphological changes in F‐actin stress fiber formation supported these results and revealed corresponding changes in thrombin‐induced gap formation consistent with the observed changes in permeability. In addition, depletion of candidate proteins also modulated thrombin‐stimulated p38 pro‐inflammatory signaling and APC‐induced AKT cytoprotective signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 bias signaling in endothelial cells.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM116597 and R35 GM127121 (J.T.), UC Tobacco‐related Disease Research Program Predoctoral Fellowship (C.C.R), and NIH/NIGMS K12 GM068524 (O.M.I., J.D.L.)

Emerging Role of Compartmentalized G Protein-Coupled Receptor Signaling in the Cardiovascular Field.

G protein-coupled receptors (GPCRs) are cell surface receptors that for many years have been considered to function exclusively at the plasma membrane, where they bind to extracellular ligands and activate G protein signaling cascades. According to the conventional model, these signaling events are rapidly terminated by β-arrestin (β-arr) recruitment to the activated GPCR resulting in signal desensitization and receptor internalization. However, during the past decade, emerging evidence suggest that many GPCRs can continue to activate G proteins from intracellular compartments after they have been internalized. G protein signaling from intracellular compartments is in general more sustained compared to G protein signaling at the plasma membrane. Notably, the particular location closer to the nucleus is beneficial for selective cellular functions such as regulation of gene transcription. Here, we review key GPCRs that undergo compartmentalized G protein signaling and discuss molecular considerations and requirements for this signaling to occur. Our main focus will be on receptors involved in the regulation of important physiological and pathological cardiovascular functions. We also discuss how sustained G protein activation from intracellular compartments may be involved in cellular functions that are distinct from functions regulated by plasma membrane G protein signaling, and the corresponding significance in cardiovascular physiology.

GPCR and Alcohol-Related Behaviors in Genetically Modified Mice.

G protein-coupled receptors (GPCRs) constitute the largest class of cell surface signaling receptors and regulate major neurobiological processes. Accordingly, GPCRs represent primary targets for the treatment of brain disorders. Several human genetic polymorphisms affecting GPCRs have been associated to different components of alcohol use disorder (AUD). Moreover, GPCRs have been reported to contribute to several features of alcohol-related behaviors in animal models. Besides traditional pharmacological tools, genetic-based approaches mostly aimed at deleting GPCR genes provided substantial information on how key GPCRs drive alcohol-related behaviors. In this review, we summarize the alcohol phenotypes that ensue from genetic manipulation, in particular gene deletion, of key GPCRs in rodents. We focused on GPCRs that belong to fundamental neuronal systems that have been shown as potential targets for the development of AUD treatment. Data are reviewed with particular emphasis on alcohol reward, seeking, and consumption which are behaviors that capture essential aspects of AUD. Literature survey indicates that in most cases, there is still a gap in defining the intracellular transducers and the functional crosstalk of GPCRs as well as the neuronal populations in which their signaling regulates alcohol actions. Further, the implication of only a few orphan GPCRs has been so far investigated in animal models. Combining advanced pharmacological technologies with more specific genetically modified animals and behavioral preclinical models is likely necessary to deepen our understanding in how GPCR signaling contributes to AUD and for drug discovery.

Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G-protein coupled receptors (GPCRs). The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance, metabolism, and the central modulation of food intake, motivation, reward, and mood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short-chain fatty acids (SCFAs), lactate, and different bacterial strains, including Bifidobacterium and Lactobacillus genera, to modulate GHSR-1a signaling. We identify, for what is to our knowledge the first time, a potent effect of microbiota-derived metabolites on GHSR-1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin-mediated signaling through the GHSR-1a. We suggest a novel route of communication between the gut microbiota and the host via modulation of GHSR-1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of the microbiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery.-Torres-Fuentes, C., Golubeva, A. V., Zhdanov, A. V., Wallace, S., Arboleya, S., Papkovsky, D. B., El Aidy, S., Ross, P., Roy, B. L., Stanton, C., Dinan, T. G., Cryan, J. F., Schellekens, H. Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

Endothelial Differentiation G Protein-Coupled Receptor 5 Plays an Important Role in Induction and Maintenance of Pluripotency.

Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318–331

Structural organization of a major neuronal G protein regulator, the RGS7-Gβ5-R7BP complex.

Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.

Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.

Characterization of GPCR signaling in hypoxia.

G protein-coupled receptors (GPCRs) signal in response to various external stimuli including stress. GPCR signaling has been shown to play a critical role in the adaptation of cell response to limited oxygen supply. Hypoxia has been implicated in cardiovascular diseases, human pulmonary arterial responses, and persistent pulmonary hypertension in newborns. One of the key GPCRs implicated in hypoxia is the prostanoid receptor, thromboxane A2 receptor (TP). Hypoxia can affect TP localization, stability, and activity both in vivo and in vitro. To elucidate hypoxia-mediated GPCR signaling in vitro, we lay out a general strategy to perform hypoxic experiments using both primary pulmonary artery smooth muscle cells and TP expressed in HEK293T cells. We describe assay for measuring moderate tissue hypoxia using static cell cultures, monitoring pericellular media oxygen content, and signaling of TP.

Activation of a Muscarinic G-Protein Coupled Receptor and Structure-Based Design of Allosteric Modulators

G-protein coupled receptors (GPCRs) are important cell signaling membrane proteins that represent primary targets of about one third of currently marketed drugs. The M2 muscarinic receptor is a key GPCR that regulates the human heart rate and contractile forces of cardiomyocytes, and is thus targeted for treating many heart diseases. Here, we have performed long-timescale accelerated molecular dynamics (aMD) simulations and captured activation of the M2 receptor at an atomistic level [1]. The receptor activation is characterized by large-scale structural rearrangements of the transmembrane helices and conformational changes in the inter-helical salt bridge and hydrogen bond interactions [2]. Furthermore, using the aMD simulation-derived structural ensembles that account for the receptor flexibility, we have mapped the receptor surface for druggable allosteric sites [3] and targeted the extracellular vestibule for designing allosteric modulators. Retrospective docking of known ligands is first carried out to validate the simulation receptor ensembles, followed by prospective docking to predict new allosteric ligands from the National Cancer Institute compound library. The computationally selected lead compounds are finally tested using experimental binding and functional assays to identify potential allosteric modulators of the M2 muscarinic GPCR [4].1. Miao, Y., et al., Activation and dynamic network of the M2 muscarinic receptor. Proc Natl Acad Sci U S A, 2013. 110(27): p. 10982-10987.2. Miao, Y., S.E. Nichols, and J.A. McCammon, Free Energy Landscape of G-Protein Coupled Receptors, Explored by Accelerated Molecular Dynamics. Physical Chemistry Chemical Physics, 2014. 16(14): p. 6398-6406.3. Miao, Y., S.E. Nichols, and J.A. McCammon, Mapping of Allosteric Druggable Sites in Activation-Associated Conformers of the M2 Muscarinic Receptor. Chemical Biology & Drug Design, 2013. 83(2): p. 237-246.4. Miao, Y., et al., Structural-Based Design of Allosteric Modulators of the M2 Muscarinic G-protein Coupled Receptor. In Preparation, 2015.

Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

Understanding the effects on constitutive activation and drug binding of a D130N mutation in the β2 adrenergic receptor via molecular dynamics simulation.

G-protein-coupled receptors (GPCRs) are currently one of the largest families of drug targets. The constitutive activation induced by mutation of key GPCR residues is associated closely with various diseases. However, the structural basis underlying such activation and its role in drug binding has remained unclear. Herein, we used all-atom molecular dynamics simulations and free energy calculations to study the effects of a D130N mutation on the structure of β2 adrenergic receptor (β2AR) and its binding of the agonist salbutamol. The results indicate that the mutation caused significant changes in some key helices. In particular, the mutation leads to the departure of transmembrane 3 (TM3) from transmembrane 6 (TM6) and marked changes in the NPxxY region as well as the complete disruption of a key ionic lock, all of which contribute to the observed constitutive activation. In addition, the D130N mutation weakens some important H-bonds, leading to structural changes in these regions. Binding free energy calculations indicate that van der Waals and electrostatic interactions are the main driving forces in binding salbutamol; however, binding strength in the mutant β2AR is significantly enhanced mainly through modifying electrostatic interactions. Further analysis revealed that the increase in binding energy upon mutation stems mainly from the H-bonds formed between the hydroxyl group of salbutamol and the serine residues of TM5. This observation suggests that modifications of the H-bond groups of this drug could significantly influence drug efficacy in the treatment of diseases associated with this mutation.

Identification of G protein-coupled receptors in Schistosoma haematobium and S. mansoni by comparative genomics.

BackgroundSchistosomiasis is a parasitic disease affecting ~200 million people worldwide. Schistosoma haematobium and S. mansoni are two relatively closely related schistosomes (blood flukes), and the causative agents of urogenital and hepatointestinal schistosomiasis, respectively. The availability of genomic, transcriptomic and proteomic data sets for these two schistosomes now provides unprecedented opportunities to explore their biology, host interactions and schistosomiasis at the molecular level. A particularly important group of molecules involved in a range of biological and developmental processes in schistosomes and other parasites are the G protein-coupled receptors (GPCRs). Although GPCRs have been studied in schistosomes, there has been no detailed comparison of these receptors between closely related species. Here, using a genomic-bioinformatic approach, we identified and characterised key GPCRs in S. haematobium and S. mansoni (two closely related species of schistosome).MethodsUsing a Hidden Markov Model (HMM) and Support Vector Machine (SVM)-based pipeline, we classified and sub-classified GPCRs of S. haematobium and S. mansoni, combined with phylogenetic and transcription analyses.ResultsWe identified and classified classes A, B, C and F as well as an unclassified group of GPCRs encoded in the genomes of S. haematobium and S. mansoni. In addition, we characterised ligand-specific subclasses (i.e. amine, peptide, opsin and orphan) within class A (rhodopsin-like).ConclusionsMost GPCRs shared a high degree of similarity and conservation, except for members of a particular clade (designated SmGPR), which appear to have diverged between S. haematobium and S. mansoni and might explain, to some extent, some of the underlying biological differences between these two schistosomes. The present set of annotated GPCRs provides a basis for future functional genomic studies of cellular GPCR-mediated signal transduction and a resource for future drug discovery efforts in schistosomes.

Free energy landscape of G-protein coupled receptors, explored by accelerated molecular dynamics.

G-protein coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. They are known to adopt multiple conformational states (e.g., inactive, intermediate and active) during their modulation of various cell signaling pathways. Here, the free energy landscape of GPCRs is explored using accelerated molecular dynamics (aMD) simulations as demonstrated on the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes. Free energy profiles of important structural motifs that undergo conformational transitions upon GPCR activation and allosteric signaling are analyzed in detail, including the Arg(3.50)-Glu(6.30) ionic lock, the Trp(6.48) toggle switch and the hydrogen interactions between Tyr(5.58)-Tyr(7.53).

Activation of the M2 Muscarinic Receptor and Computer-Aided Design of Receptor-Selective Allosteric Drugs

G-protein coupled receptors (GPCRs) are important cell signaling membrane proteins that have been targeted by ∼30-40% of marketed drugs for treating many human diseases including cancer and heart failure. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined in an inactive antagonist-bound state. Through accelerated molecular dynamics (aMD) enhanced sampling simulation, we captured activation of the M2 receptor that occurs on the millisecond timescales(1). The receptor activation is characterized by large-scale structural rearrangements of the transmembrane helices via an intermediate state. With activation-associated conformers of the M2 receptor revealed from aMD simulation, a fragment-based site mapping program FTMAP is applied to explore the receptor surface. Seven allosteric sites are identified with two distributed in the solvent-exposed extracellular and intracellular mouth regions and five in the lipid-exposed pockets formed by transmembrane helices of the receptor(2). Virtual screening is then performed to select small-molecule drugs that bind these allosteric sites with differential affinity and thus stabilize the receptor in the corresponding conformational states. In contrast to highly conserved residues in the orthosteric site where endogenous ligands bind, residues in the allosteric sites exhibit large diversity across different GPCR subtypes. Therefore, ligands that bind GPCR allosteric sites are able to provide potential receptor-selective therapeutics.

Activation and dynamic network of the M2 muscarinic receptor

G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist-bound state. Here, activation of the M2 receptor is directly observed via accelerated molecular dynamics simulation, in contrast to previous microsecond-timescale conventional molecular dynamics simulations in which the receptor remained inactive. Receptor activation is characterized by formation of a Tyr206(5.58)-Tyr440(7.53) hydrogen bond and ∼6-Å outward tilting of the cytoplasmic end of transmembrane α-helix 6, preceded by relocation of Trp400(6.48) toward Phe195(5.47) and Val199(5.51) and flipping of Tyr430(7.43) away from the ligand-binding cavity. Network analysis reveals that communication in the intracellular domains is greatly weakened during activation of the receptor. Together with the finding that residue motions in the ligand-binding and G-protein-coupling sites of the apo receptor are correlated, this result highlights a dynamic network for allosteric regulation of the M2 receptor activation.

Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy

Cardiac cells, like those of the other tissues, undergo regulation through membrane-bound proteins known as G protein-coupled receptors (GPCRs). β-adrenergic receptors (βARs) are key GPCRs expressed on cardiomyocytes and their role is crucial in cardiac physiology since they regulate inotropic and chronotropic responses of the sympathetic nervous system (SNS). In compromised conditions such as heart failure (HF), chronic βAR hyperstimulation occurs via SNS activation resulting in receptor dysregulation and down-regulation and consequently there is a marked reduction of myocardial inotropic reserve and continued loss of pump function. Data accumulated over the last two decades indicates that a primary culprit in initiating and maintain βAR dysfunction in the injured and stressed heart is GPCR kinase 2 (GRK2), which was originally known as βARK1 (for βAR kinase). GRK2 is up-regulated in the failing heart due to chronic SNS activity and targeting this kinase has emerged as a novel therapeutic strategy in HF. Indeed, its inhibition or genetic deletion in several disparate animal models of HF including a pre-clinical pig model has shown that GRK2 targeting improves functional and morphological parameters of the failing heart. Moreover, non-βAR properties of GRK2 appear to also contribute to its pathological effects and thus, its inhibition will likely complement existing therapies such as βAR blockade. This review will explore recent research regarding GRK2 inhibition; in particular it will focus on the GRK2 inhibitor peptide known as βARKct, which represents new hope in the treatment against HF progression.

Cholesterol Modulates the Membrane Effects and Spatial Organization of Membrane-Penetrating Ligands for G-Protein Coupled Receptors

The ligands of certain G-protein coupled receptors (GPCRs) reach their target from the lipid bilayer. We have probed the organization and dynamics of a such lipid bilayer-penetrating ligand, the endogeneous ligand for kappa-opioid receptor (KOR) dynorphin A (1-17) (DynA), using molecular dynamics (MD) simulations of DynA in cholesterol-depleted and cholesterol-enriched model membranes. DynA penetrates deep inside fluid dimyristoylphosphatidylcholine (DMPC) bilayers, and resides with its N-terminal helix at ∼6Å away from the bilayer mid-plane, in a tilted orientation, at ∼50°C angle with respect to membrane normal. In contrast, inside DMPC/Chol membranes with 20% cholesterol (DMPC/Chol) the DynA is situated ∼5Å higher, i.e. closer to the lipid/water interface and in a relatively vertical orientation. Compared to the DMPC/Chol membranes, the DMPC membrane shows greater thinning around the insertion and permits a stronger influx of water inside the hydrocarbon. Relating these results to data about key GPCR residues that have been implicated in interactions with membrane-inserting GPCR ligands, we conclude that the position of DynA correlates with generally proposed GPCR ligand entry pathways in DMPC/Chol, but not in pure DMPC. Our predictions provide a possible mechanistic explanation as to why DynA binding to KOR, and the subsequent activation of the receptor, is facilitated in cholesterol-enriched environments. To obtain a quantitative description of DynA-induced membrane deformations we used a continuum theory of membrane deformations (CTMD) that is based on hydrophobic matching. Comparison with the MD results suggests that the lipid tail packing energy makes a significant contribution to predicting equilibrium membrane shape around DynA in the DMPC/Chol mixtures. This energy term was therefore introduced in the CTMD framework, thereby extending the applicability of the CTMD to quantify membrane remodeling by multi-helical proteins.