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Sample records for predicted g-protein-coupled receptor

  1. Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

    PubMed Central

    Kmiecik, Sebastian; Jamroz, Michal; Kolinski, Michal

    2014-01-01

    G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs. PMID:24896119

  2. Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

    PubMed

    Cabrera, Ilva E; Pacentine, Itallia V; Lim, Andrew; Guerrero, Nayeli; Krystofova, Svetlana; Li, Liande; Michkov, Alexander V; Servin, Jacqueline A; Ahrendt, Steven R; Carrillo, Alexander J; Davidson, Liza M; Barsoum, Andrew H; Cao, Jackie; Castillo, Ronald; Chen, Wan-Ching; Dinkchian, Alex; Kim, Stephanie; Kitada, Sho M; Lai, Taffani H; Mach, Ashley; Malekyan, Cristin; Moua, Toua R; Torres, Carlos Rojas; Yamamoto, Alaina; Borkovich, Katherine A

    2015-10-13

    G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.

  3. Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

    PubMed

    Cabrera, Ilva E; Pacentine, Itallia V; Lim, Andrew; Guerrero, Nayeli; Krystofova, Svetlana; Li, Liande; Michkov, Alexander V; Servin, Jacqueline A; Ahrendt, Steven R; Carrillo, Alexander J; Davidson, Liza M; Barsoum, Andrew H; Cao, Jackie; Castillo, Ronald; Chen, Wan-Ching; Dinkchian, Alex; Kim, Stephanie; Kitada, Sho M; Lai, Taffani H; Mach, Ashley; Malekyan, Cristin; Moua, Toua R; Torres, Carlos Rojas; Yamamoto, Alaina; Borkovich, Katherine A

    2015-12-01

    G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization. PMID:26464358

  4. GPCR-MPredictor: multi-level prediction of G protein-coupled receptors using genetic ensemble.

    PubMed

    Naveed, Muhammad; Khan, Asifullah; Khan, Asif Ullah

    2012-05-01

    G protein-coupled receptors (GPCRs) are transmembrane proteins, which transduce signals from extracellular ligands to intracellular G protein. Automatic classification of GPCRs can provide important information for the development of novel drugs in pharmaceutical industry. In this paper, we propose an evolutionary approach, GPCR-MPredictor, which combines individual classifiers for predicting GPCRs. GPCR-MPredictor is a web predictor that can efficiently predict GPCRs at five levels. The first level determines whether a protein sequence is a GPCR or a non-GPCR. If the predicted sequence is a GPCR, then it is further classified into family, subfamily, sub-subfamily, and subtype levels. In this work, our aim is to analyze the discriminative power of different feature extraction and classification strategies in case of GPCRs prediction and then to use an evolutionary ensemble approach for enhanced prediction performance. Features are extracted using amino acid composition, pseudo amino acid composition, and dipeptide composition of protein sequences. Different classification approaches, such as k-nearest neighbor (KNN), support vector machine (SVM), probabilistic neural networks (PNN), J48, Adaboost, and Naives Bayes, have been used to classify GPCRs. The proposed hierarchical GA-based ensemble classifier exploits the prediction results of SVM, KNN, PNN, and J48 at each level. The GA-based ensemble yields an accuracy of 99.75, 92.45, 87.80, 83.57, and 96.17% at the five levels, on the first dataset. We further perform predictions on a dataset consisting of 8,000 GPCRs at the family, subfamily, and sub-subfamily level, and on two other datasets of 365 and 167 GPCRs at the second and fourth levels, respectively. In comparison with the existing methods, the results demonstrate the effectiveness of our proposed GPCR-MPredictor in classifying GPCRs families. It is accessible at http://111.68.99.218/gpcr-mpredictor/.

  5. Prediction of G Protein-Coupled Receptors with SVM-Prot Features and Random Forest.

    PubMed

    Liao, Zhijun; Ju, Ying; Zou, Quan

    2016-01-01

    G protein-coupled receptors (GPCRs) are the largest receptor superfamily. In this paper, we try to employ physical-chemical properties, which come from SVM-Prot, to represent GPCR. Random Forest was utilized as classifier for distinguishing them from other protein sequences. MEME suite was used to detect the most significant 10 conserved motifs of human GPCRs. In the testing datasets, the average accuracy was 91.61%, and the average AUC was 0.9282. MEME discovery analysis showed that many motifs aggregated in the seven hydrophobic helices transmembrane regions adapt to the characteristic of GPCRs. All of the above indicate that our machine-learning method can successfully distinguish GPCRs from non-GPCRs. PMID:27529053

  6. Prediction of G Protein-Coupled Receptors with SVM-Prot Features and Random Forest

    PubMed Central

    Ju, Ying

    2016-01-01

    G protein-coupled receptors (GPCRs) are the largest receptor superfamily. In this paper, we try to employ physical-chemical properties, which come from SVM-Prot, to represent GPCR. Random Forest was utilized as classifier for distinguishing them from other protein sequences. MEME suite was used to detect the most significant 10 conserved motifs of human GPCRs. In the testing datasets, the average accuracy was 91.61%, and the average AUC was 0.9282. MEME discovery analysis showed that many motifs aggregated in the seven hydrophobic helices transmembrane regions adapt to the characteristic of GPCRs. All of the above indicate that our machine-learning method can successfully distinguish GPCRs from non-GPCRs. PMID:27529053

  7. Predicting G-protein-coupled receptors families using different physiochemical properties and pseudo amino acid composition.

    PubMed

    Rehman, Zia-Ur; Mirza, Muhammad Tayyeb; Khan, Asifullah; Xhaard, Henri

    2013-01-01

    G-protein-coupled receptors (GPCRs) initiate signaling pathways via trimetric guanine nucleotide-binding proteins. GPCRs are classified based on their ligand-binding properties and molecular phylogenetic analyses. Nonetheless, these later analyses are in most case dependent on multiple sequence alignments, themselves dependent on human intervention and expertise. Alignment-free classifications of GPCR sequences, in addition to being unbiased, present many applications uncovering hidden physicochemical parameters shared among specific groups of receptors, to being used in automated workflows for large-scale molecular modeling applications. Current alignment-free classification methods, however, do not reach a full accuracy. This chapter discusses how GPCRs amino acid sequences can be classified using pseudo amino acid composition and multiscale energy representation of different physiochemical properties of amino acids. A hybrid feature extraction strategy is shown to be suitable to represent GPCRs and to be able to exploit GPCR amino acid sequence discrimination capability in spatial as well as transform domain. Classification strategies such as support vector machine and probabilistic neural network are then discussed in regards to GPCRs classification. The work of GPCR-Hybrid web predictor is also discussed.

  8. Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cattle tick, Rhipicephalus (Boophilus) microplus, is a pest which causes multiple health complications in cattle. The G-protein coupled receptor (GPCR) super-family presents an interesting target for developing novel tick control methods. However, GPCRs share limited sequence similarity among or...

  9. G Protein-Coupled Receptors in Cancer.

    PubMed

    Bar-Shavit, Rachel; Maoz, Myriam; Kancharla, Arun; Nag, Jeetendra Kumar; Agranovich, Daniel; Grisaru-Granovsky, Sorina; Uziely, Beatrice

    2016-01-01

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of "cancer driver" GPCRs. Emerging data on GPCR biology point to functional selectivity and "biased agonism"; hence, there is a diminishing enthusiasm for the concept of "one drug per GPCR target" and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics. PMID:27529230

  10. G Protein-Coupled Receptors in Cancer

    PubMed Central

    Bar-Shavit, Rachel; Maoz, Myriam; Kancharla, Arun; Nag, Jeetendra Kumar; Agranovich, Daniel; Grisaru-Granovsky, Sorina; Uziely, Beatrice

    2016-01-01

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics. PMID:27529230

  11. Signaling through G protein coupled receptors

    PubMed Central

    2009-01-01

    Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role. PMID:19826234

  12. A novel fractal approach for predicting G-protein-coupled receptors and their subfamilies with support vector machines.

    PubMed

    Nie, Guoping; Li, Yong; Wang, Feichi; Wang, Siwen; Hu, Xuehai

    2015-01-01

    G-protein-coupled receptors (GPCRs) are seven membrane-spanning proteins and regulate many important physiological processes, such as vision, neurotransmission, immune response and so on. GPCRs-related pathways are the targets of a large number of marketed drugs. Therefore, the design of a reliable computational model for predicting GPCRs from amino acid sequence has long been a significant biomedical problem. Chaos game representation (CGR) reveals the fractal patterns hidden in protein sequences, and then fractal dimension (FD) is an important feature of these highly irregular geometries with concise mathematical expression. Here, in order to extract important features from GPCR protein sequences, CGR algorithm, fractal dimension and amino acid composition (AAC) are employed to formulate the numerical features of protein samples. Four groups of features are considered, and each group is evaluated by support vector machine (SVM) and 10-fold cross-validation test. To test the performance of the present method, a new non-redundant dataset was built based on latest GPCRDB database. Comparing the results of numerical experiments, the group of combined features with AAC and FD gets the best result, the accuracy is 99.22% and Matthew's correlation coefficient (MCC) is 0.9845 for identifying GPCRs from non-GPCRs. Moreover, if it is classified as a GPCR, it will be further put into the second level, which will classify a GPCR into one of the five main subfamilies. At this level, the group of combined features with AAC and FD also gets best accuracy 85.73%. Finally, the proposed predictor is also compared with existing methods and shows better performances.

  13. patGPCR: A Multitemplate Approach for Improving 3D Structure Prediction of Transmembrane Helices of G-Protein-Coupled Receptors

    PubMed Central

    Wu, Hongjie; Lü, Qiang; Quan, Lijun; Qian, Peide; Xia, Xiaoyan

    2013-01-01

    The structures of the seven transmembrane helices of G-protein-coupled receptors are critically involved in many aspects of these receptors, such as receptor stability, ligand docking, and molecular function. Most of the previous multitemplate approaches have built a “super” template with very little merging of aligned fragments from different templates. Here, we present a parallelized multitemplate approach, patGPCR, to predict the 3D structures of transmembrane helices of G-protein-coupled receptors. patGPCR, which employs a bundle-packing related energy function that extends on the RosettaMem energy, parallelizes eight pipelines for transmembrane helix refinement and exchanges the optimized helix structures from multiple templates. We have investigated the performance of patGPCR on a test set containing eight determined G-protein-coupled receptors. The results indicate that patGPCR improves the TM RMSD of the predicted models by 33.64% on average against a single-template method. Compared with other homology approaches, the best models for five of the eight targets built by patGPCR had a lower TM RMSD than that obtained from SWISS-MODEL; patGPCR also showed lower average TM RMSD than single-template and multiple-template MODELLER. PMID:23554839

  14. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    DOEpatents

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  15. G-protein-coupled receptors in intestinal chemosensation.

    PubMed

    Reimann, Frank; Tolhurst, Gwen; Gribble, Fiona M

    2012-04-01

    Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and peripheral nutrient disposal. G-protein-coupled receptors responsive to a range of nutrients and other food components have been identified, and many are localized to intestinal chemosensory cells, eliciting hormonal and neuronal signaling to the brain and periphery. This review examines the role of G-protein-coupled receptors as signaling molecules in the gut, with a particular focus on pathways relevant to appetite and glucose homeostasis. PMID:22482725

  16. Molecular pharmacology of G protein-coupled receptors.

    PubMed

    Summers, R J

    2016-10-01

    This themed issue of the British Journal of Pharmacology stems from the eighth in the series of meetings on the Molecular Pharmacology of G protein coupled receptors (MPGPCR) held as part of a joint meeting with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) in Melbourne Australia from 7 to 11 December 2014. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc. PMID:27682321

  17. Molecular pharmacology of G protein-coupled receptors.

    PubMed

    Summers, R J

    2016-10-01

    This themed issue of the British Journal of Pharmacology stems from the eighth in the series of meetings on the Molecular Pharmacology of G protein coupled receptors (MPGPCR) held as part of a joint meeting with the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) in Melbourne Australia from 7 to 11 December 2014. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  18. Applications of molecular replacement to G protein-coupled receptors

    SciTech Connect

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-11-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

  19. G Protein-Coupled Receptors in Anopheles gambiae

    NASA Astrophysics Data System (ADS)

    Hill, Catherine A.; Fox, A. Nicole; Pitts, R. Jason; Kent, Lauren B.; Tan, Perciliz L.; Chrystal, Mathew A.; Cravchik, Anibal; Collins, Frank H.; Robertson, Hugh M.; Zwiebel, Laurence J.

    2002-10-01

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  20. G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation

    PubMed Central

    Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

    2016-01-01

    The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand—receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

  1. Olfactory receptors: G protein-coupled receptors and beyond.

    PubMed

    Spehr, Marc; Munger, Steven D

    2009-06-01

    Sensing the chemical environment is critical for all organisms. Diverse animals from insects to mammals utilize highly organized olfactory system to detect, encode, and process chemostimuli that may carry important information critical for health, survival, social interactions and reproduction. Therefore, for animals to properly interpret and react to their environment it is imperative that the olfactory system recognizes chemical stimuli with appropriate selectivity and sensitivity. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. In the nearly two decades since the discovery that the mammalian odorant receptor gene family constitutes the largest group of G protein-coupled receptor (GPCR) genes, much attention has been focused on the roles of GPCRs in vertebrate and invertebrate olfaction. However, is has become clear that the 'family' of olfactory receptors is highly diverse, with roles for enzymes and ligand-gated ion channels as well as GPCRs in the primary detection of olfactory stimuli. PMID:19383089

  2. Molecular signatures of G-protein-coupled receptors.

    PubMed

    Venkatakrishnan, A J; Deupi, Xavier; Lebon, Guillaume; Tate, Christopher G; Schertler, Gebhard F; Babu, M Madan

    2013-02-14

    G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine. PMID:23407534

  3. Desensitization of G protein-coupled receptors and neuronal functions.

    PubMed

    Gainetdinov, Raul R; Premont, Richard T; Bohn, Laura M; Lefkowitz, Robert J; Caron, Marc G

    2004-01-01

    G protein-coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology. Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions. GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein-coupled receptor kinases (GRKs). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and beta arrestins in major brain functions. In particular, screening of genetically modified mice lacking individual GRKs or beta arrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and mu-opioid receptor regulation of locomotion and analgesia. An important and specific role of GRKs and beta arrestins in regulating physiological responsiveness to psychostimulants and morphine suggests potential involvement of these molecules in certain brain disorders, such as addiction, Parkinson's disease, mood disorders, and schizophrenia. Furthermore, the utility of a pharmacological strategy aimed at targeting this GPCR desensitization machinery to regulate brain functions can be envisaged. PMID:15217328

  4. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    PubMed

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-08-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.

  5. Biacore analysis with stabilized G-protein-coupled receptors.

    PubMed

    Rich, Rebecca L; Errey, James; Marshall, Fiona; Myszka, David G

    2011-02-15

    Using stabilized forms of β₁ adrenergic and A₂(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.

  6. Serial femtosecond crystallography datasets from G protein-coupled receptors

    PubMed Central

    White, Thomas A.; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A.; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R.; Yoon, Chun Hong; Yefanov, Oleksandr M.; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E.; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  7. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    PubMed

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  8. Novel Allosteric Modulators of G Protein-coupled Receptors*

    PubMed Central

    Gentry, Patrick R.; Sexton, Patrick M.; Christopoulos, Arthur

    2015-01-01

    G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization. PMID:26100627

  9. Crystal Structure of a Lipid G Protein-Coupled Receptor

    SciTech Connect

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  10. Application of BRET for studying G protein-coupled receptors.

    PubMed

    Kaczor, Agnieszka A; Makarska-Bialokoz, Magdalena; Selent, Jana; de la Fuente, Rocío A; Martí-Solano, Maria; Castro, Marián

    2014-05-01

    G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

  11. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  12. Serial femtosecond crystallography of G protein-coupled receptors.

    PubMed

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J; Koglin, Jason E; Seibert, M Marvin; Wang, Chong; Shah, Syed T A; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A; Beyerlein, Kenneth R; White, Thomas A; Chapman, Henry N; Caffrey, Martin; Spence, John C H; Stevens, Raymond C; Cherezov, Vadim

    2013-12-20

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.

  13. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  14. Serial femtosecond crystallography of G protein-coupled receptors.

    PubMed

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J; Koglin, Jason E; Seibert, M Marvin; Wang, Chong; Shah, Syed T A; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A; Beyerlein, Kenneth R; White, Thomas A; Chapman, Henry N; Caffrey, Martin; Spence, John C H; Stevens, Raymond C; Cherezov, Vadim

    2013-12-20

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  15. Classification of G-protein coupled receptors at four levels.

    PubMed

    Gao, Qing-Bin; Wang, Zheng-Zhi

    2006-11-01

    G-protein coupled receptors (GPCRs) are transmembrane proteins which via G-proteins initiate some of the important signaling pathways in a cell and are involved in various physiological processes. Thus, computational prediction and classification of GPCRs can supply significant information for the development of novel drugs in pharmaceutical industry. In this paper, a nearest neighbor method has been introduced to discriminate GPCRs from non-GPCRs and subsequently classify GPCRs at four levels on the basis of amino acid composition and dipeptide composition of proteins. Its performance is evaluated on a non-redundant dataset consisted of 1406 GPCRs for six families and 1406 globular proteins using the jackknife test. The present method based on amino acid composition achieved an overall accuracy of 96.4% and Matthew's correlation coefficient (MCC) of 0.930 for correctly picking out the GPCRs from globular proteins. The overall accuracy and MCC were further enhanced to 99.8% and 0.996 by dipeptide composition-based method. On the other hand, the present method has successfully classified 1406 GPCRs into six families with an overall accuracy of 89.6 and 98.8% using amino acid composition and dipeptide composition, respectively. For the subfamily prediction of 1181 GPCRs of rhodopsin-like family, the present method achieved an overall accuracy of 76.7 and 94.5% based on the amino acid composition and dipeptide composition, respectively. Finally, GPCRs belonging to the amine subfamily and olfactory subfamily of rhodopsin-like family were further analyzed at the type level. The overall accuracy of dipeptide composition-based method for the classification of amine type and olfactory type of GPCRs reached 94.5 and 86.9%, respectively, while the overall accuracy of amino acid composition-based method was very low for both subfamilies. In comparison with existing methods in the literature, the present method also displayed great competitiveness. These results demonstrate

  16. Genetic approaches to unraveling G protein-coupled receptor biology.

    PubMed

    Aparicio, Samuel A J R; Powell, Justin

    2004-09-01

    Genetic approaches to validating G protein-coupled receptors (GPCRs) have proven to be a powerful research tool, especially knockout studies in rodents. To date, data related to in vivo function have been published on approximately half of the human rhodopsin-like family-1 GPCRs, which can be attributed to the use of mouse knockouts. It is likely that many currently unknown yet important therapeutic mechanisms will be uncovered through knockout screens in mice. One such recent discovery is the elucidation of the in vivo function of the GPCR GPR54 through mouse genetics, and its subsequent validation in human populations. Although previously unsuspected, GPR54 has been found to be a master-regulator of the hypothalamic-pituitary-gonadal axis.

  17. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  18. Lysophospholipids and their G protein-coupled receptors in atherosclerosis.

    PubMed

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2016-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc. PMID:26709762

  19. G protein-coupled receptors in regulation of body weight.

    PubMed

    Schiöth, Helgi B

    2006-06-01

    In this issue of CNS & Neurological Disorders-Drug Targets, we focus on G protein-coupled receptors (GPCRs) that are involved in regulating body weight. In six reviews, the melanocortins system (including MC4 and MC3 receptors, Agrp, MSH), the NPY receptors (including NPY-Y1, NPY-Y2, and NPY-Y5, PYY3-36), the cannabinoid system (including the development of rimonabant), the ghrelin (GHS, growth hormone secretagogue) system, the monoamine GPCRs (including serotonin, adrenergic and histamine receptors), orexin (hypocretin) system and the galanin receptors are covered. In this overview, an introduction to the GPCRs and the field of central regulation of food intake is provided together with brief mentioning of some other GPCRs that are also implicated in regulation of body weight, such as the melanin-concentrating hormone (MCH), neuromedin U, prolactin-releasing peptide (PrRP), bombesin, cholecystokinin (CCK), Glucagon-like peptide-1 (GLP-1) (and oxyntomodulin), neuropeptide B (NPB) and neuropeptide W (NPW), opioids peptides, free fatty acid (FFA) receptors (GPR40, GPR41). In total over 40 GPCRs are listed that have been implicated to affect regulation of body weight.

  20. Heterologous expression of G-protein-coupled receptors in yeast.

    PubMed

    Bertheleme, Nicolas; Singh, Shweta; Dowell, Simon; Byrne, Bernadette

    2015-01-01

    Heterologous yeast expression systems have been successfully used for the production of G-protein-coupled receptors (GPCRs) for both structural and functional studies. Yeast combine comparatively low cost and short culture times with straightforward generation of expression clones. They also perform some key posttranslational modifications not possible in bacterial systems. There are two major yeast expression systems, Pichia pastoris and Saccharomyces cerevisiae, both of which have been used for the production of GPCRs. P. pastoris has a proven track record for the production of large amounts of GPCR for structural studies. High-resolution crystal structures of both the adenosine A2A and the histamine H1 receptors have been obtained using protein expressed in this system. S. cerevisiae is relatively easy to engineer and this has resulted in the development of sophisticated tools for the functional characterization of GPCRs. In this chapter, we provide protocols for both large-scale receptor expression in P. pastoris for structural studies and small-scale receptor expression in S. cerevisiae for functional characterization. In both cases, the receptor used is the human adenosine A2A receptor. The results that both we and others have obtained using these protocols show the wide utility of the yeast expression systems for the production of GPCRs.

  1. Model Organisms in G Protein-Coupled Receptor Research.

    PubMed

    Langenhan, Tobias; Barr, Maureen M; Bruchas, Michael R; Ewer, John; Griffith, Leslie C; Maiellaro, Isabella; Taghert, Paul H; White, Benjamin H; Monk, Kelly R

    2015-09-01

    The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism. PMID:25979002

  2. Structure and Function of Serotonin G protein Coupled Receptors

    PubMed Central

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  3. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    NASA Astrophysics Data System (ADS)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  4. Heterodimerization and Surface Localization of G Protein Coupled Receptors

    PubMed Central

    Minneman, Kenneth P.

    2007-01-01

    G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: 1) How many functionally and pharmacologically distinct receptor subtypes exist in vivo? 2) Which GPCRs physically associate, and in what stochiometries? 3) What are the roles of individual subunits in binding ligand and activating responses? 4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease. PMID:17011524

  5. Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors.

    PubMed

    Thompson, Dawn; Pusch, Margareta; Whistler, Jennifer L

    2007-10-01

    After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.

  6. Stabilization of G protein-coupled receptors by point mutations

    PubMed Central

    Heydenreich, Franziska M.; Vuckovic, Ziva; Matkovic, Milos; Veprintsev, Dmitry B.

    2015-01-01

    G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs. PMID:25941489

  7. Discovery of three novel orphan G-protein-coupled receptors.

    PubMed

    Marchese, A; Sawzdargo, M; Nguyen, T; Cheng, R; Heng, H H; Nowak, T; Im, D S; Lynch, K R; George, S R; O'dowd, B F

    1999-02-15

    We have discovered three novel human genes, GPR34, GPR44, and GPR45, encoding family A G-protein-coupled receptors (GPCRs). The receptor encoded by GPR34 is most similar to the P2Y receptor subfamily, while the receptor encoded by GPR44 is most similar to chemoattractant receptors. The receptor encoded by GPR45 is the mammalian orthologue of a putative lysophosphatidic acid receptor from Xenopus laevis. Partial sequence of GPR34 was discovered during a search of the GenBank database of expressed sequence tags (ESTs). This sequence information was used both to isolate the full-length translational open reading frame from a human genomic library and to assemble a contig from additional GPR34 EST cDNAs. Northern blot and in situ hybridization analyses revealed GPR34 mRNA transcripts in several human and rat brain regions. Also, we used polymerase chain reaction (PCR) to amplify human genomic DNA using degenerate oligonucleotides designed from sequences encoding transmembrane domains 3 and 7 of opioid and somatostatin receptors. Two PCR products partially encoding novel GPCRs, named GPR44 and GPR45, were discovered and used to isolate the full-length translational open reading frames from a human genomic library. Both GPR44 and GPR45 are expressed in the central nervous system and periphery. For chromosomal localization, fluorescence in situ hybridization analysis was performed to assign GPR34 to chromosomes 4p12 and Xp11. 3, GPR44 to chromosome 11q12-q13.3, and GPR45 to chromosome 2q11. 1-q12. PMID:10036181

  8. Surface plasmon resonance applied to G protein-coupled receptors

    PubMed Central

    Locatelli-Hoops, Silvia; Yeliseev, Alexei A.; Gawrisch, Klaus; Gorshkova, Inna

    2013-01-01

    G protein-coupled receptors (GPCR) are integral membrane proteins that transmit signals from external stimuli to the cell interior via activation of GTP-binding proteins (G proteins) thereby mediating key sensorial, hormonal, metabolic, immunological, and neurotransmission processes. Elucidating their structure and mechanism of interaction with extracellular and intracellular binding partners is of fundamental importance and highly relevant to rational design of new effective drugs. Surface plasmon resonance (SPR) has become a method of choice for studying biomolecular interactions at interfaces because measurements take place in real-time and do not require labeling of any of the interactants. However, due to the particular challenges imposed by the high hydrophobicity of membrane proteins and the great diversity of receptor-stimulating ligands, the application of this technique to characterize interactions of GPCR is still in the developmental phase. Here we give an overview of the principle of SPR and analyze current approaches for the preparation of the sensor chip surface, capture and stabilization of GPCR, and experimental design to characterize their interaction with ligands, G proteins and specific antibodies. PMID:24466506

  9. Snapin interacts with G-protein coupled receptor PKR2.

    PubMed

    Song, Jian; Li, Jie; Liu, Hua-die; Liu, Wei; Feng, Yong; Zhou, Xiao-Tao; Li, Jia-Da

    2016-01-15

    Mutations in Prokineticin receptor 2 (PKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, characterized by delayed puberty and infertility. In this study, we performed yeast two-hybrid screening by using PKR2 C-terminus (amino acids 333-384) as a bait, and identified Snapin as a novel interaction partner for PKR2. The interaction of Snapin and PKR2 was confirmed in GST pull-down and co-immunoprecipitation studies. We further demonstrated that two α-helix domains in Snapin are required for the interaction. And the interactive motifs of PKR2 were mapped to YFK (343-345) and HWR (351-353), which shared a similar sequence of two aromatic amino acids followed by a basic amino acid. Disruption of Snapin-PKR2 interaction did not affect PKR2 signaling, but increased the ligand-induced degradation, implying a role of Snapin in the trafficking of PKR2.

  10. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

  11. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    PubMed Central

    Gao, Xin; Wu, Tongyu; Johnson, Kirby D.; Lahvic, Jamie L.; Ranheim, Erik A.; Zon, Leonard I.; Bresnick, Emery H.

    2016-01-01

    Summary Hematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis. PMID:26905203

  12. Alpha-Bulges in G Protein-Coupled Receptors

    PubMed Central

    van der Kant, Rob; Vriend, Gert

    2014-01-01

    Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs) that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process. PMID:24806342

  13. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling.

    PubMed

    Zhao, Wen-Li; Wang, Di; Liu, Chun-Yan; Zhao, Xiao-Fan

    2016-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism. PMID:27412951

  14. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling

    PubMed Central

    Zhao, Wen-Li; Wang, Di; Liu, Chun-Yan; Zhao, Xiao-Fan

    2016-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism. PMID:27412951

  15. G protein coupled receptors as targets for next generation pesticides.

    PubMed

    Audsley, Neil; Down, Rachel E

    2015-12-01

    There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion

  16. Conformational Fluctuations in G-Protein-Coupled Receptors

    NASA Astrophysics Data System (ADS)

    Brown, Michael F.

    2014-03-01

    G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

  17. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  18. A ligand channel through the G protein coupled receptor opsin.

    PubMed

    Hildebrand, Peter W; Scheerer, Patrick; Park, Jung Hee; Choe, Hui-Woog; Piechnick, Ronny; Ernst, Oliver P; Hofmann, Klaus Peter; Heck, Martin

    2009-01-01

    The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through

  19. Tools for investigating functional interactions between ligands and G-protein-coupled receptors.

    PubMed

    Lerner, M R

    1994-04-01

    A general assay for evaluating functional interactions between ligands and G-protein-coupled receptors within minutes has been developed. The system uses the principles employed by animals such as reptiles, amphibians and fish to control their colors. In nature, activation of G-protein-coupled receptors expressed by skin cells called chromatophores effects pigment redistribution within the cells to change an animal's coloration. The in vitro 'chameleon in a dish' equivalent can use essentially any cloned G-protein-coupled receptor. PMID:7517590

  20. High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139

    PubMed Central

    Wang, Jia; Zhu, Lin-yun; Liu, Qing; Hentzer, Morten; Smith, Garrick Paul; Wang, Ming-wei

    2015-01-01

    Aim: To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules. Methods: Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies. Results: Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds. Conclusion: The findings provide important tools for further study of this orphan G-protein coupled receptor. PMID:26027661

  1. A dual receptor crosstalk model of G-protein-coupled signal transduction.

    PubMed

    Flaherty, Patrick; Radhakrishnan, Mala L; Dinh, Tuan; Rebres, Robert A; Roach, Tamara I; Jordan, Michael I; Arkin, Adam P

    2008-09-26

    Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Galphai-coupled C5a receptor and the Galphaq-coupled UDP receptor signaling systems that results in synergistic calcium release.

  2. Computational methods for studying G protein-coupled receptors (GPCRs).

    PubMed

    Kaczor, Agnieszka A; Rutkowska, Ewelina; Bartuzi, Damian; Targowska-Duda, Katarzyna M; Matosiuk, Dariusz; Selent, Jana

    2016-01-01

    The functioning of GPCRs is classically described by the ternary complex model as the interplay of three basic components: a receptor, an agonist, and a G protein. According to this model, receptor activation results from an interaction with an agonist, which translates into the activation of a particular G protein in the intracellular compartment that, in turn, is able to initiate particular signaling cascades. Extensive studies on GPCRs have led to new findings which open unexplored and exciting possibilities for drug design and safer and more effective treatments with GPCR targeting drugs. These include discovery of novel signaling mechanisms such as ligand promiscuity resulting in multitarget ligands and signaling cross-talks, allosteric modulation, biased agonism, and formation of receptor homo- and heterodimers and oligomers which can be efficiently studied with computational methods. Computer-aided drug design techniques can reduce the cost of drug development by up to 50%. In particular structure- and ligand-based virtual screening techniques are a valuable tool for identifying new leads and have been shown to be especially efficient for GPCRs in comparison to water-soluble proteins. Modern computer-aided approaches can be helpful for the discovery of compounds with designed affinity profiles. Furthermore, homology modeling facilitated by a growing number of available templates as well as molecular docking supported by sophisticated techniques of molecular dynamics and quantitative structure-activity relationship models are an excellent source of information about drug-receptor interactions at the molecular level. PMID:26928552

  3. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    PubMed

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

  4. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  5. GPCRdb: the G protein-coupled receptor database - an introduction.

    PubMed

    Munk, C; Isberg, V; Mordalski, S; Harpsøe, K; Rataj, K; Hauser, A S; Kolb, P; Bojarski, A J; Vriend, G; Gloriam, D E

    2016-07-01

    GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality. PMID:27155948

  6. GPCRdb: the G protein-coupled receptor database - an introduction.

    PubMed

    Munk, C; Isberg, V; Mordalski, S; Harpsøe, K; Rataj, K; Hauser, A S; Kolb, P; Bojarski, A J; Vriend, G; Gloriam, D E

    2016-07-01

    GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality.

  7. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  8. Deletion of G-protein-coupled receptor 55 promotes obesity by reducing physical activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cannabinoid receptor 1 (CB1) is the best-characterized cannabinoid receptor, and CB1 antagonists are used in clinical trials to treat obesity. Because of the wide range of CB1 functions, the side effects of CB1 antagonists pose serious concerns. G-protein-coupled receptor 55 (GPR55) is an atypical c...

  9. Exploring the Biology of G Protein-Coupled Receptors from In Vitro to In Vivo.

    PubMed

    Bohn, Laura M; Lohse, Martin J; Nitabach, Michael N; Taghert, Paul H; Smit, Martine J

    2015-09-01

    In August 2014, an international group of researchers gathered for 5 days at the Lorentz Center in Leiden, The Netherlands, to explore the technical and conceptual issues associated with the analysis of G protein-coupled receptor functions utilizing information from crystal structure models to the use of model organisms. This collection of review articles evolved from the 5-day meeting, with brief presentations and structured discussion periods that were designed to identify key questions remaining in understanding G protein-coupled receptor function and to propose novel strategies by integrating scientific disciplines to guide future research.

  10. Alternative Splicing of G Protein-Coupled Receptors: Relevance to Pain Management.

    PubMed

    Oladosu, Folabomi A; Maixner, William; Nackley, Andrea G

    2015-08-01

    Drugs that target G protein-coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse effects associated with these drugs. Variability in drug responses is due, in part, to individuals' diversity in alternative splicing of pain-relevant GPCRs. G protein-coupled receptor alternative splice variants often exhibit distinct tissue distribution patterns, drug-binding properties, and signaling characteristics that may impact disease pathology as well as the extent and direction of analgesic effects. We review the importance of GPCRs and their known splice variants to the management of pain.

  11. Use of Designer G Protein-Coupled Receptors to Dissect Metabolic Pathways.

    PubMed

    Wess, Jürgen

    2016-09-01

    G protein-coupled receptors (GPCRs) regulate virtually all metabolic processes, including glucose and energy homeostasis. Recently, the use of designer GPCRs referred to as designer receptors exclusively activated by designer drug (DREADDs) has made it possible to dissect metabolically relevant GPCR signaling pathways in a temporally and spatially controlled fashion in vivo. PMID:27381463

  12. In silico prediction of the G-protein coupled receptors expressed during the metamorphic molt of Sagmariasus verreauxi (Crustacea: Decapoda) by mining transcriptomic data: RNA-seq to repertoire.

    PubMed

    Buckley, Sean J; Fitzgibbon, Quinn P; Smith, Gregory G; Ventura, Tomer

    2016-03-01

    Against a backdrop of food insecurity, the farming of decapod crustaceans is a rapidly expanding and globally significant source of food protein. Sagmariasus verreauxi spiny lobster, the subject of this study, are decapods of underdeveloped aquaculture potential. Crustacean neuropeptide G-protein coupled receptors (GPCRs) mediate endocrine pathways that are integral to animal fecundity, growth and survival. The potential use of novel biotechnologies to enhance GPCR-mediated physiology may assist in improving the health and productivity of farmed decapod populations. This study catalogues the GPCRs expressed in the early developmental stages, as well as adult tissues, with a view to illuminating key neuropeptide receptors. De novo assembled contiguous sequences generated from transcriptomic reads of metamorphic and post metamorphic S. verreauxi were filtered for seven transmembrane domains, and used as a reference for iterative re-mapping. Subsequent putative GPCR open reading frames (ORFs) were BLAST annotated, categorised, and compared to published orthologues based on phylogenetic analysis. A total of 85 GPCRs were digitally predicted, that represented each of the four arthropod subfamilies. They generally displayed low-level and non-differential metamorphic expression with few exceptions that we examined using RT-PCR and qPCR. Two putative CHH-like neuropeptide receptors were annotated. Three dimensional structural modelling suggests that these receptors exhibit a conserved extracellular ligand binding pocket, providing support to the notion that these receptors co-evolved with their ligands across Decapoda. This perhaps narrows the search for means to increase productivity of farmed decapod populations.

  13. In silico prediction of the G-protein coupled receptors expressed during the metamorphic molt of Sagmariasus verreauxi (Crustacea: Decapoda) by mining transcriptomic data: RNA-seq to repertoire.

    PubMed

    Buckley, Sean J; Fitzgibbon, Quinn P; Smith, Gregory G; Ventura, Tomer

    2016-03-01

    Against a backdrop of food insecurity, the farming of decapod crustaceans is a rapidly expanding and globally significant source of food protein. Sagmariasus verreauxi spiny lobster, the subject of this study, are decapods of underdeveloped aquaculture potential. Crustacean neuropeptide G-protein coupled receptors (GPCRs) mediate endocrine pathways that are integral to animal fecundity, growth and survival. The potential use of novel biotechnologies to enhance GPCR-mediated physiology may assist in improving the health and productivity of farmed decapod populations. This study catalogues the GPCRs expressed in the early developmental stages, as well as adult tissues, with a view to illuminating key neuropeptide receptors. De novo assembled contiguous sequences generated from transcriptomic reads of metamorphic and post metamorphic S. verreauxi were filtered for seven transmembrane domains, and used as a reference for iterative re-mapping. Subsequent putative GPCR open reading frames (ORFs) were BLAST annotated, categorised, and compared to published orthologues based on phylogenetic analysis. A total of 85 GPCRs were digitally predicted, that represented each of the four arthropod subfamilies. They generally displayed low-level and non-differential metamorphic expression with few exceptions that we examined using RT-PCR and qPCR. Two putative CHH-like neuropeptide receptors were annotated. Three dimensional structural modelling suggests that these receptors exhibit a conserved extracellular ligand binding pocket, providing support to the notion that these receptors co-evolved with their ligands across Decapoda. This perhaps narrows the search for means to increase productivity of farmed decapod populations. PMID:26850661

  14. Activation of family C G-protein-coupled receptors by the tripeptide glutathione.

    PubMed

    Wang, Minghua; Yao, Yi; Kuang, Donghui; Hampson, David R

    2006-03-31

    The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the gamma-aminobutyric acid, type B (GABAB) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acid-activated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or co-ligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [3H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wild-type and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABAB receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions. PMID:16455645

  15. An algebra of dimerization and its implications for G-protein coupled receptor signaling.

    PubMed

    Woolf, Peter J; Linderman, Jennifer J

    2004-07-21

    Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.

  16. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    PubMed Central

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  17. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    PubMed

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  18. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response*

    PubMed Central

    Alvaro, Christopher G.; Thorner, Jeremy

    2016-01-01

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeast Saccharomyces cerevisiae were isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. PMID:26907689

  19. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana.

    PubMed

    Josefsson, L G; Rask, L

    1997-10-15

    We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-protein-coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G-protein-coupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to cAMP receptors from the slime mold Dictyostelium discoideum. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the so-called rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and cAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved

  20. Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function

    PubMed Central

    Gomes, Ivone; Gupta, Achla; Bushlin, Ittai; Devi, Lakshmi A.

    2014-01-01

    Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects. PMID:25520661

  1. A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers.

    PubMed

    Waldhoer, Maria; Fong, Jamie; Jones, Robert M; Lunzer, Mary M; Sharma, Shiv K; Kostenis, Evi; Portoghese, Philip S; Whistler, Jennifer L

    2005-06-21

    There has been much speculation regarding the functional relevance of G protein-coupled receptor heterodimers, primarily because demonstrating their existence in vivo has proven to be a considerable challenge. Here we show that the opioid agonist ligand 6'-guanidinonaltrindole (6'-GNTI) has the unique property of selectively activating only opioid receptor heterodimers but not homomers. Importantly, 6'-GNTI is an analgesic, thereby demonstrating that opioid receptor heterodimers are indeed functionally relevant in vivo. However, 6'-GNTI induces analgesia only when it is administered in the spinal cord but not in the brain, suggesting that the organization of heterodimers is tissue-specific. This study demonstrates a proof of concept for tissue-selective drug targeting based on G protein-coupled receptor heterodimerization. Importantly, targeting opioid heterodimers could provide an approach toward the design of analgesic drugs with reduced side effects.

  2. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

    PubMed Central

    Strittmatter, S M; Cannon, S C; Ross, E M; Higashijima, T; Fishman, M C

    1993-01-01

    The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. Images Fig. 1 Fig. 2 PMID:7685122

  3. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases

    PubMed Central

    Singh, Anukriti; Nunes, Jessica J.; Ateeq, Bushra

    2015-01-01

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases. PMID:25981295

  4. Structure-based drug design for G protein-coupled receptors.

    PubMed

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. PMID:24418607

  5. Conducting the G-protein Coupled Receptor (GPCR) Signaling Symphony in Cardiovascular Diseases: New Therapeutic Approaches.

    PubMed

    Belmonte, Stephen L; Blaxall, Burns C

    2012-01-01

    G protein-coupled receptors (GPCRs) are a virtually ubiquitous class of membrane-bound receptors, which functionally couple hormone or neurotransmitter signals to physiological responses. Dysregulation of GPCR signaling contributes to the pathophysiology of a host of cardiovascular disorders. Pharmacological agents targeting GPCRs have been established as therapeutic options for decades. Nevertheless, the persistent burden of cardiovascular diseases necessitates improved treatments. To that end, exciting drug development efforts have begun to focus on novel compounds that discriminately activate particular GPCR signaling pathways.

  6. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

    PubMed

    Busato, Mirko; Giorgetti, Alejandro

    2016-08-01

    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well.

  7. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

    PubMed

    Busato, Mirko; Giorgetti, Alejandro

    2016-08-01

    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well. PMID:27102413

  8. New roles of G protein-coupled receptor kinase 2 (GRK2) in cell migration

    PubMed Central

    Penela, Petronila; Ribas, Catalina; Aymerich, Ivette

    2009-01-01

    G protein-coupled receptor kinase 2 (GRK2) was initially identified as a key player, together with β-arrestins, in the regulation of multiple G protein-coupled receptors (GPCR). Further research has revealed a complex GRK2 interactome, that includes a variety of proteins related to cell motility, and a role for GRK2 kinase activity in inhibiting chemokine-induced immune cell migration. In addition, we have recently reported that GRK2 positively regulates integrin and sphingosine-1-phosphate-dependent motility in epithelial cell types and fibroblasts, acting as a scaffold molecule. We suggest that the positive or negative correlation of GRK2 levels with cell migration would depend on the cell type, specific stimuli acting through plasma membrane receptors, or on the signalling context, leading to differential networks of interaction of GRK2 with cell migration-related signalosomes. PMID:19372742

  9. Identification of human OGR1, a novel G protein-coupled receptor that maps to chromosome 14

    SciTech Connect

    Xu, Yan; Casey, G.

    1996-07-15

    We describe the cloning of a novel G protein-coupled receptor, termed ovarian cancer G protein-coupled receptor 1 (OGR1), from an ovarian cancer cell line that maps to chromosome 14q31. The predicted open reading frame of OGR1 encodes a protein of 365 amino acids. OGR1 is expressed as a single 3.0-kb transcript in several tissues, including spleen, testis, small intestine, peripheral blood leukocytes, brain, heart, lung, placenta, and kidney, with no detectable OGR1 expression in thymus, prostate, ovary, colon, liver, skeletal muscle, or pancreas. OGR1 shares strongest homology (49-54%) with the human orphan receptor GPR4. The structural features of OGR1 suggest that the ligand for OGR1 is likely to be a small peptide or a small bioactive molecule. 25 refs., 3 figs.

  10. G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

    PubMed Central

    Connelly, Timothy; Yu, Yiqun; Grosmaitre, Xavier; Wang, Jue; Santarelli, Lindsey C.; Savigner, Agnes; Qiao, Xin; Wang, Zhenshan; Storm, Daniel R.; Ma, Minghong

    2015-01-01

    Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction. PMID:25550517

  11. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

  12. An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

    PubMed

    Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

    2005-04-29

    We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

  13. Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

    PubMed Central

    Scearce-Levie, Kimberly; Lieberman, Michael D; Elliott, Heather H; Conklin, Bruce R

    2005-01-01

    Background The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of Gi-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. Results Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. Conclusions These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic Gi-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute Gi signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo. PMID:15707483

  14. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    PubMed Central

    Wellendorph, P; Bräuner-Osborne, H

    2009-01-01

    Family C of human G-protein-coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two γ-aminobutyric acid type B (GABAB1–2) subunits forming the heterodimeric GABAB receptor, the calcium-sensing receptor, three taste1 receptors (T1R1–3), a promiscuous L-α-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABAB1–2 and T1R2–3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity and the initial steps in receptor activation. PMID:19298394

  15. A novel subgroup of class I G-protein-coupled receptors.

    PubMed

    Schöneberg, T; Schulz, A; Grosse, R; Schade, R; Henklein, P; Schultz, G; Gudermann, T

    1999-07-01

    Based on structural similarities of an expressed sequence tag with the platelet-activating factor (PAF) receptor a cDNA clone encoding a novel G-protein-coupled receptor (GPCR), named GPR34, was isolated from a human fetal brain cDNA library. Genomic DNA analyses revealed the receptor to be encoded by an intronless single-copy gene at Xp11. 3-11.4. The predicted 381-amino-acid protein disclosed all structural features characteristic of a member of the class I GPCR family. Except an obvious sequence homology in transmembrane domain 6, no further similarities to the PAF receptor or any other known GPCR were found. The corresponding mouse receptor DNA was isolated from a genomic P1 library displaying a 90% amino acid identity compared to the human receptor. Phylogenetic studies showed that GPR34 is preserved among vertebrates, and the existence of GPR34 subtypes was demonstrated. The receptor mRNA is abundantly expressed in human and mouse tissues. In addition to the major 2-kb transcript, a 4-kb transcript was found only in mouse liver and testis. Expression of the human GPR34 in COS-7 cells followed by Western blot studies revealed specific bands of a highly glycosylated protein between 75 and 90 kDa. A number of potential ligands including phospholipids, leukotrienes, hydroxy-eicosatetraenoic acids, nucleotides and peptides were tested in functional assays. Since none of the applied substances led to significant changes in second messenger levels (cAMP and inositol phosphates), the natural ligand and coupling profile of this novel GPCR subgroup remains unknown. PMID:10395919

  16. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

  17. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  18. Cloning and characterization of additional members of the G protein-coupled receptor family.

    PubMed

    Lee, D K; Lynch, K R; Nguyen, T; Im, D S; Cheng, R; Saldivia, V R; Liu, Y; Liu, I S; Heng, H H; Seeman, P; George, S R; O'Dowd, B F; Marchese, A

    2000-02-29

    A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

  19. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium.

    PubMed

    Plested, C P; Bernal, A L

    2001-03-01

    G-protein coupled receptors (GPCRs) are key to maintaining uterine quiescence and inducing phasic contractions at term. However, the biochemical mechanisms whereby uterine GPCRs are desensitised and re-sensitised during these physiological conditions are unknown. For example, the number of oxytocin receptors (OTRs) on uterine myocytes decrease significantly after the addition of oxytocin. Therefore, further understanding of the desensitisation/re-sensitisation of the OTR and other uterine GPCRs during pregnancy may provide a target for more efficient tocolytic drugs and more selective ways to modulate uterine activity. Here, we briefly review some of the mechanisms that may be involved during OTR and other GPCR desensitisation. Experimental Physiology (2001) 86.2, 303-312.

  20. Functional characterization of G-protein-coupled receptors: a bioinformatics approach.

    PubMed

    Tovo-Rodrigues, L; Roux, A; Hutz, M H; Rohde, L A; Woods, A S

    2014-09-26

    Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCR's intra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic and charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, may be critical for the nervous system's functional plasticity.

  1. Large-scale production and protein engineering of G protein-coupled receptors for structural studies

    PubMed Central

    Milić, Dalibor; Veprintsev, Dmitry B.

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures. PMID:25873898

  2. Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

    PubMed

    Milić, Dalibor; Veprintsev, Dmitry B

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

  3. G-Protein Coupled Receptor Resensitization – Appreciating the Balancing Act of Receptor Function

    PubMed Central

    Mohan, Maradumane L.; Vasudevan, Neelakantan T.; Gupta, Manveen K.; Martelli, Elizabeth E.; Prasad, Sathyamangla V. Naga

    2015-01-01

    G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used β-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms. PMID:22697395

  4. Constitutive Dimerization of the G-Protein Coupled Receptor, Neurotensin Receptor 1, Reconstituted into Phospholipid Bilayers

    PubMed Central

    Harding, Peter J.; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H.; Smith, Eleanor; Armitage, Judith P.; Watts, Anthony

    2009-01-01

    Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed. PMID:19186134

  5. Diversity and Impact of Rare Variants in Genes Encoding the Platelet G Protein-Coupled Receptors

    PubMed Central

    Jones, Matthew L.; Norman, Jane E.; Morgan, Neil V.; Mundell, Stuart J.; Lordkipanidzé, Marie; Lowe, Gillian C.; Daly, Martina E.; Simpson, Michael A.; Drake, Sian; Watson, Steve P.

    2015-01-01

    Summary Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF ≥ 1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes. PMID:25567036

  6. Signaling, physiological functions and clinical relevance of the G protein-coupled estrogen receptor GPER

    PubMed Central

    Prossnitz, Eric R.; Barton, Matthias

    2009-01-01

    GPR30, now named GPER1 (G protein-coupled estrogen receptor1) or GPER here, was first identified as an orphan 7- transmembrane G protein-coupled receptor by multiple laboratories using either homology cloning or differential expression and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. The actions of estrogen are extensive in the body and are thought to be mediated predominantly by classical nuclear estrogen receptors that act as transcription factors/regulators. Nevertheless, certain aspects of estrogen function remain incompatible with the generally accepted mechanisms of classical estrogen receptor action. Many recent studies have revealed that GPER contributes to some of the actions of estrogen, including rapid signaling events and rapid transcriptional activation. With the introduction of GPER-selective ligands and GPER knockout mice, the functions of GPER are becoming more clearly defined. In many cases, there appears to be a complex interplay between the two receptor systems, suggesting that estrogen-mediated physiological responses may be mediated by either receptor or a combination of both receptor types, with important medical implications. PMID:19442754

  7. Increased G Protein-Coupled Receptor Kinase (GRK) Expression in the Anterior Cingulate Cortex in Schizophrenia

    PubMed Central

    Funk, Adam J.; Haroutunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

    2014-01-01

    Background Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that expression of GRK proteins are altered in schizophrenia, consistent with previous findings of alterations up and downstream from this family of molecules that facilitate intracellular signaling processes. Methods In this study we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (N = 36) and a comparison group (N = 33). To control for antipsychotic treatment we measured these same targets in haloperidol treated vs. untreated rats (N = 10 for both). Results We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. Conclusion These data suggest that increased GRK5 expression may contribute the the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. PMID:25153362

  8. Understanding the added value of g-protein-coupled receptor heteromers.

    PubMed

    Franco, Nuria; Franco, Rafael

    2014-01-01

    G-protein-coupled receptors (GPCRs) constitute the most populated family of proteins within the human genome. Since the early sixties work on GPCRs and on GPCR-mediated signaling has led to a number of awards, the most recent being the Nobel Prize in Chemistry for 2012. The future of GPCRs research is surely based on their capacity for heteromerization. Receptor heteromers offer a series of challenges that will help in providing success in academic/basic research and translation into more effective and safer drugs.

  9. The Concise Guide to Pharmacology 2013/14: G Protein-Coupled Receptors

    PubMed Central

    Alexander, Stephen PH; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Spedding, Michael; Peters, John A; Harmar, Anthony J

    2013-01-01

    The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. G protein-coupled receptors are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates. PMID:24517644

  10. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

    PubMed Central

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. PMID:26861299

  11. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling.

    PubMed

    Riaz, Anjum; Huang, Ying; Johansson, Staffan

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)-AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K-AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted. PMID:26861299

  12. Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

    PubMed Central

    Parker, Michael S.; Sah, Renu; Balasubramaniam, Ambikaipakan; Park, Edwards A.; Sallee, Floyd R.; Parker, Steven L.

    2014-01-01

    The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists. PMID:24651459

  13. Monitoring endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

    PubMed Central

    Tower-Gilchrist, Cristy; Styers, Melanie L.; Yoder, Bradley K.; Berbari, Nicolas F.; Sztul, Elizabeth

    2016-01-01

    Endocytic trafficking of G protein-coupled receptors (GPCRs) regulates the number of cell surface receptors available for activation by agonists and serves as one mechanism that controls the intensity and duration of signaling. Deregulation of GPCR-mediated signaling pathways results in a multitude of diseases, and thus extensive efforts have been directed toward understand the pathways and molecular events that regulate endocytic trafficking of these receptors. The general paradigms associated with internalization and recycling, as well as many of the key regulators involved in endosomal trafficking of GPCRs have been identified. This knowledge provides goalposts to facilitate the analysis of endosomal pathways traversed by previously uncharacterized GPCRs. Some of the most informative markers associated with GPCR transit are the Rab members of the Ras-related family of small GTPases. Individual Rabs show high selectivity for distinct endosomal compartments, and thus co-localization of a GPCR with a particular Rab informs on the internalization pathway traversed by the receptor. Progress in our knowledge of endosomal trafficking of GPCRs has been achieved through advances in our ability to tag GPCRs and Rabs with fluorescent proteins and perform live cell imaging of multiple fluorophores, allowing real-time observation of receptor trafficking between subcellular compartments in a cell culture model. PMID:24359959

  14. Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

    NASA Astrophysics Data System (ADS)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2016-02-01

    This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

  15. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization.

    PubMed

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor-receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery.

  16. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization.

    PubMed

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor-receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  17. Allosteric mechanisms of G protein coupled receptor signaling: a structural perspective

    PubMed Central

    Thaker, Tarjani M.; Kaya, Ali I.; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T.M.

    2012-01-01

    G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways. PMID:22052489

  18. Alternative Splicing of G-protein Coupled Receptors: Relevance to Pain Management

    PubMed Central

    Oladosu, Folabomi A.; Maixner, William; Nackley, Andrea G.

    2015-01-01

    Drugs that target G-protein coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse side effects associated with these drugs. Variability in drug responses is, in part, due to individuals’ diversity in alternative splicing of pain-relevant GPCRs. GPCR alternative splice variants often exhibit distinct tissue distribution patterns, drug binding properties, and signaling characteristics that may impact disease pathology as well as the size and direction of analgesic effects. Here, we review the importance of GPCRs and their known splice variants to the management of pain. PMID:26250730

  19. Signaling and regulation of G protein-coupled receptors in airway smooth muscle

    PubMed Central

    Billington, Charlotte K; Penn, Raymond B

    2003-01-01

    Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state. PMID:12648290

  20. Aptamer BC 007 - A broad spectrum neutralizer of pathogenic autoantibodies against G-protein-coupled receptors.

    PubMed

    Haberland, Annekathrin; Holtzhauer, Martin; Schlichtiger, Alice; Bartel, Sabine; Schimke, Ingolf; Müller, Johannes; Dandel, Michael; Luppa, Peter B; Wallukat, Gerd

    2016-10-15

    The effect of autoantibodies on G-protein coupled receptors in the pathogenesis of diseases, especially of the heart and vascular system, is an increasingly accepted fact today. Dilated cardiomyopathy (DCM) is the most intensively investigated pathological situation of these. With DCM, autoantibodies against the β1-adrenoceptor and the muscarinic M2-receptor have been found in high percentage of investigated patients. Immunoadsorption for autoantibody removal has already shown a long-term beneficial therapeutic effect, but has remained limited in its application because of the complexity of this method. A new easy applicable treatment strategy has, therefore, been discovered. Because of intra- and inter-loop epitope variability of the β1-adrenoceptor specific autoantibodies and also the occurrence of further autoantibodies of this class such as the ones against the β2- and α1-adrenoceptor, the ETA-, proteinase activated-, and the AT1-receptors in different pathological situations, this newly discovered broad-spectrum neutralizer of all these autoantibodies - aptamer BC 007 - is under development. The binding and neutralizing effect was investigated applying a bioassay of spontaneously beating neonatal rat cardiomyocytes and enzyme-linked immunosorbent assay (ELISA) - technology. The usefulness of aptamer BC 007 to specify column technology for the removal of serum autoantibodies was also demonstrated. The presented data suggest that aptamer BC 007 might be an appropriate molecule candidate to support future research about the meaning of G-protein-coupled receptor autoantibodies.

  1. The origin and evolution of G protein-coupled receptor kinases.

    PubMed

    Mushegian, Arcady; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2012-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals.

  2. The Origin and Evolution of G Protein-Coupled Receptor Kinases

    PubMed Central

    Mushegian, Arcady; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2012-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals. PMID:22442725

  3. Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules

    PubMed Central

    Camp, Nathan D; Lee, Kyung-Soon; Wacker-Mhyre, Jennifer L; Kountz, Timothy S; Park, Ji-Min; Harris, Dorathy-Ann; Estrada, Marianne; Stewart, Aaron; Wolf-Yadlin, Alejandro; Hague, Chris

    2015-01-01

    Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)—a key regulator of cardiovascular, urinary and CNS function—binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces. PMID:26617989

  4. Molecular evolution of a chordate specific family of G protein-coupled receptors

    PubMed Central

    2011-01-01

    Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become

  5. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions.

    PubMed

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala; Peisajovich, Sergio G

    2016-08-04

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease.

  6. Molecular basis for activation of G protein-coupled receptor kinases

    SciTech Connect

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  7. Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1.

    PubMed Central

    Zondag, G C; Postma, F R; Etten, I V; Verlaan, I; Moolenaar, W H

    1998-01-01

    Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct G-protein-coupled receptors to evoke similar responses, including Ca2+ mobilization, adenylate cyclase inhibition, and mitogen-activated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1/Edg-2) is similar to an orphan Gi-coupled receptor termed Edg-1. Here we show that expression of Edg-1 in Sf9 and COS-7 cells results in inhibition of adenylate cyclase and activation of MAP kinase (Gi-mediated), but not Ca2+ mobilization, in response to S1P. These responses are specific in that (i) S1P action is not mimicked by LPA, and (ii) Vzg-1/Edg-2 cannot substitute for Edg-1. Thus the Edg-1 receptor is capable of mediating a subset of the cellular responses to S1P. PMID:9480864

  8. Biased ligands at G-protein-coupled receptors: promise and progress.

    PubMed

    Violin, Jonathan D; Crombie, Aimee L; Soergel, David G; Lark, Michael W

    2014-07-01

    Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the μ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development.

  9. Biased ligands at G-protein-coupled receptors: promise and progress.

    PubMed

    Violin, Jonathan D; Crombie, Aimee L; Soergel, David G; Lark, Michael W

    2014-07-01

    Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the μ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development. PMID:24878326

  10. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    PubMed Central

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  11. Heteromerization of G Protein-Coupled Receptors: Relevance to Neurological Disorders and Neurotherapeutics

    PubMed Central

    Albizu, Laura; Moreno, José L.; González-Maeso, Javier; Sealfon, Stuart C.

    2011-01-01

    Because G protein-coupled receptors (GPCRs) are numerous, widely expressed and involved in major physiological responses, they represent a relevant therapeutic target for drug discovery, particularly regarding pharmacological treatments of neurological disorders. Among the biological phenomena regulating receptor function, GPCR heteromerization is an important emerging area of interest and investigation. There is increasing evidence showing that heteromerization contributes to the pharmacological heterogeneity of GPCRs by modulating receptor ontogeny, activation and recycling. Although in many cases the physiological relevance of receptor heteromerization has not been fully established, the unique pharmacological and functional properties of heteromers are likely to lead to new strategies in clinical medicine. This review describes the main GPCR heteromers and their implications for major neurological disorders such as Parkinson’s disease, schizophrenia and addiction. A better understanding of molecular mechanisms underlying drug interactions related to the targeting of receptor heteromers could provide more specific and efficient therapeutic agents for the treatment of brain diseases. PMID:20632964

  12. Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

    PubMed Central

    Salom, David; Cao, Pengxiu; Sun, Wenyu; Kramp, Kristopher; Jastrzebska, Beata; Jin, Hui; Feng, Zhaoyang; Palczewski, Krzysztof

    2012-01-01

    New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A2A subtype receptor [(h)A2AR] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6–1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A2AR were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.—Salom, D., Cao, P., Sun, W., Kramp, K., Jastrzebska, B., Jin, H., Feng, Z., Palczewski, K. Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans. PMID:22090314

  13. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    SciTech Connect

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K.

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  14. Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

    PubMed

    Le Gouill, C; Parent, J L; Caron, C A; Gaudreau, R; Volkov, L; Rola-Pleszczynski, M; Stanková, J

    1999-04-30

    Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases. PMID:10212233

  15. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    PubMed Central

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. PMID:27314054

  16. G-protein-coupled receptor signaling and the EGF network in endocrine systems.

    PubMed

    Hsieh, Minnie; Conti, Marco

    2005-09-01

    The epidermal growth factor (EGF) network is composed of a complex array of growth factors synthesized as precursors and expressed on the cell surface. These latent growth factors are activated by cleavage and shedding from the cell surface and act by binding to various homo- and hetero-dimers of the EGF receptors (ErbBs). Although the exact molecular steps are poorly understood, ligand binding to G-protein-coupled receptors as diverse as the beta-adrenoceptors or the lysophosphatidic acid receptors leads to shedding of EGF growth factors and activation of EGF receptors. Recent observations from the pituitary and in the ovary are providing new insight into the role of this network in endocrine systems.

  17. Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

    PubMed

    Casiraghi, Marina; Damian, Marjorie; Lescop, Ewen; Point, Elodie; Moncoq, Karine; Morellet, Nelly; Levy, Daniel; Marie, Jacky; Guittet, Eric; Banères, Jean-Louis; Catoire, Laurent J

    2016-09-01

    Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

  18. Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2

    PubMed Central

    Beautrait, Alexandre; Michalski, Kevin R.; Lopez, Thomas S.; Mannix, Katelynn M.; McDonald, Devin J.; Cutter, Amber R.; Medina, Christopher B.; Hebert, Aaron M.; Francis, Charnelle J.; Bouvier, Michel; Tesmer, John J. G.; Sterne-Marr, Rachel

    2014-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. PMID:25049229

  19. G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

    PubMed Central

    Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

    2008-01-01

    Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

  20. β-Arrestin-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy in Vivo

    PubMed Central

    Gesty-Palmer, Diane; Yuan, Ling; Martin, Bronwen; Wood, William H.; Lee, Mi-Hye; Janech, Michael G.; Tsoi, Lam C.; Zheng, W. Jim; Maudsley, Stuart

    2013-01-01

    Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, d-Trp12,Tyr34-bPTH(7–34) [bPTH(7–34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7–34) and the conventional agonist hPTH(1–34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7–34) or hPTH(1–34). Treatment of wild-type mice with bPTH(7–34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in β-arrestin2-null mice, identifying them as downstream targets of β-arrestin2-mediated signaling. In contrast, hPTH(1–34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1–34) actions were less dependent on β-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7–34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not β-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7–34) and hPTH(1–34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy. PMID:23315939

  1. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    PubMed

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

  2. G Protein-Coupled Receptor Heteromerization: A Role in Allosteric Modulation of Ligand BindingS⃞

    PubMed Central

    Gomes, Ivone; IJzerman, Adriaan P.; Ye, Kai; Maillet, Emeline L.

    2011-01-01

    It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study, we examined this possibility by using an established model system of a receptor heteromer consisting of μ and δ opioid receptors. We examined the effect of a number of μ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled δ receptor agonist, [3H]deltorphin II. We also examined the effect of δ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled μ receptor agonist, [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin ([3H]DAMGO). We show that μ receptor ligands are capable of allosterically enhancing δ receptor radioligand binding and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity. PMID:21415307

  3. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  4. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors.

    PubMed

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-09-15

    G protein-coupled receptors are divided into three classes (A, B and C) based on homology of their seven transmembrane domains. Class C is the smallest class with 22 human receptor subtypes including eight metabotropic glutamate (mGlu1-8) receptors, two GABAB receptors (GABAB1 and GABAB2), three taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we review the existence of post-translational modifications in class C G protein-coupled receptors and their regulatory roles, with particular focus on glycosylation, phosphorylation, ubiquitination, SUMOylation, disulphide bonding and lipidation.

  5. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  6. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  7. Beclin 2 Functions in Autophagy, Degradation of G Protein-Coupled Receptors, and Metabolism

    PubMed Central

    He, Congcong; Wei, Yongjie; Sun, Kai; Li, Binghua; Dong, Xiaonan; Zou, Zhongju; Liu, Yang; Kinch, Lisa N.; Khan, Shaheen; Sinha, Sangita; Xavier, Ramnik J.; Grishin, Nick V.; Xiao, Guanghua; Eskelinen, Eeva-Liisa; Scherer, Philipp E.; Whistler, Jennifer L.; Levine, Beth

    2013-01-01

    Summary The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a novel converging regulator of autophagy and GPCR turnover, and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking and metabolism. PMID:23954414

  8. Deciphering the Code to Aminergic G-Protein Coupled Receptor Drug Design

    PubMed Central

    Tan, Edwin S.; Groban, Eli S.; Jacobson, Matthew P.; Scanlan, Thomas S.

    2009-01-01

    Summary The trace amine-associated receptor 1 (TAAR1) is a biogenic amine G-protein coupled receptor (GPCR) that is potently activated by 3-iodothyronamine (1, T1AM) in vitro. Compound 1 is an endogenous derivative of the thyroid hormone thyroxine that rapidly induces hypothermia, anergia, and bradycardia when administered to mice. To explore the role of TAAR1 in mediating the effects of 1, we rationally designed and synthesized rat TAAR1 superagonists and lead antagonists using the rotamer toggle switch model of aminergic GPCR activation. The functional activity of a ligand was found to be correlated to the nature of its interactions with the rotamer switch residues. Allowing the rotamer switch residues to toggle to their active conformation was associated with agonism while interfering with this conformational transition resulted in antagonism. These agonist and antagonist design principles provide a conceptual model for understanding the relationship between the molecular structure of a drug and its pharmacological properties. PMID:18420141

  9. G-protein-coupled receptors and their (Bio) chemical significance win 2012 Nobel Prize in Chemistry.

    PubMed

    Lin, Hsi-Hsien

    2013-01-01

    G-protein-coupled receptors (GPCRs) are seven transmembrane cell surface proteins specialized in cellular communication. These receptors represent a major gateway through which cells convert external cues into intracellular signals and respond with appropriate actions. While the effects of hormones, neurotransmitters, and drugs on cells, tissues, organs, and even whole organisms are well described, the molecular identity of the direct targets and the diverse signaling mechanisms of these biological ligands have been slow and hard to define. The Nobel Prize in Chemistry for the year 2012 acknowledges the importance of GPCRs in these processes, especially for the contribution of Profs Robert J. Lefkowitz and Brian K. Kobilka to the studies of GPCRs. In this brief review, the seminal works accomplished by the two GPCR pioneers are summarized and the (bio) chemical significance of GPCRs in health and disease is discussed.

  10. G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

    PubMed Central

    Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; Booz, Valeria; Bruzzone, Santina; Sturla, Laura; Di Bona, Melody; Pesce, Mattia; Usai, Cesare; De Flora, Antonio; Zocchi, Elena

    2016-01-01

    Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation. PMID:27222287

  11. Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors.

    PubMed

    Flower, Darren R; Attwood, Teresa K

    2004-12-01

    G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein. The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics. In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods. BLAST and FastA offer broad brush strokes. Motif-based search methods add the fine detail. Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding. Together, these different views provide a more informative and a more detailed picture of GPCR structure and function. Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow. PMID:15561589

  12. From G Protein-coupled Receptor Structure Resolution to Rational Drug Design*

    PubMed Central

    Jazayeri, Ali; Dias, Joao M.; Marshall, Fiona H.

    2015-01-01

    A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design. PMID:26100628

  13. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  14. Molecular Modelling of G Protein-Coupled Receptors Through the Web.

    PubMed

    Rodríguez, David; Bello, Xabier; Gutiérrez-de-Terán, Hugo

    2012-05-01

    With the recent crystallization of several G Protein-Coupled receptors (GPCRs), homology modelling and all atom molecular dynamics (MD) simulations have proven their usefulness for exploring the structure and function of this superfamily of membrane receptors. Subsequently, automated computational protocols have been implemented as web-based servers in the recent years to produce reliable models of GPCRs, providing partial or global solutions for the structural characterization and molecular simulation of GPCRs. These dedicated modelling services represent an attractive tool for the broader community of public researchers and pharmaceutical companies, in order to assist in the structure-based drug design of GPCRs. We here collect and analyze the existing web servers, among which a previously unreported service, GPCR-ModSim, offers for the first time full atom MD simulations in the pipeline for GPCR molecular modelling. PMID:27477263

  15. The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

    PubMed Central

    Penela, Petronila; Murga, Cristina; Ribas, Catalina; Lafarga, Vanesa; Mayor, Federico

    2010-01-01

    GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist-regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context-specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2. PMID:20590581

  16. Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology.

    PubMed

    DeWire, Scott M; Violin, Jonathan D

    2011-07-01

    Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by biased ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, biased ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of biased ligand research, the current understanding of how biased ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of biased ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of biased ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.

  17. Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4.

    PubMed

    Zhu, K; Baudhuin, L M; Hong, G; Williams, F S; Cristina, K L; Kabarowski, J H; Witte, O N; Xu, Y

    2001-11-01

    Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor 1 (OGR1) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR1 (51%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF10A cells. These effects are insensitive to treatment with BN52021, WEB-2170, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = 159 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not sphingosine 1-phosphate (S1P), PAF, psychosine (Psy), glucosyl-beta1'1-sphingosine (Glu-Sph), galactosyl-beta1'1-ceramide (Gal-Cer), or lactosyl-beta1'1-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a

  18. Biased signaling through G-protein-coupled PROKR2 receptors harboring missense mutations.

    PubMed

    Sbai, Oualid; Monnier, Carine; Dodé, Catherine; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2014-08-01

    Various missense mutations in the gene coding for prokineticin receptor 2 (PROKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome. However, the functional consequences of these mutations on the different signaling pathways of this receptor have not been studied. We first showed that the wild-type PROKR2 can activate different G-protein subtypes (Gq, Gs, and Gi/o) and recruit β-arrestins in transfected HEK-293 cells. We then examined, for each of these signaling pathways, the effects of 9 mutations that did not significantly impair cell surface targeting or ligand binding of the receptor. Four mutant receptors showing defective Gq signaling (R85C, R85H, R164Q, and V331M) could still recruit β-arrestins on ligand activation, which may cause biased signaling in vivo. Conversely, the R80C receptor could activate the 3 types of G proteins but could not recruit β-arrestins. Finally, the R268C receptor could recruit β-arrestins and activate the Gq and Gs signaling pathways but could not activate the Gi/o signaling pathway. Our results validate the concept that mutations in the genes encoding membrane receptors can bias downstream signaling in various ways, possibly leading to pathogenic and, perhaps in some cases, protective (e.g., R268C) effects.

  19. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    PubMed Central

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  20. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors.

    PubMed Central

    Böhm, S K; Grady, E F; Bunnett, N W

    1997-01-01

    The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neuro-transmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and down-regulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from G-proteins. Agonist-induced receptor endocytosis also contributes to desensitization by depleting the cell surface of high-affinity receptors, and recycling of internalized receptors contributes to resensitization of cellular responses. Receptor down-regulation is a form of desensitization that occurs during continuous, long-term exposure of cells to receptor agonists. Down-regulation, which may occur during the development of drug tolerance, is characterized by depletion of the cellular receptor content, and is probably mediated by alterations in the rates of receptor degradation and synthesis. These regulatory mechanisms are important, as they govern the ability of cells to respond to agonists. A greater understanding of the mechanisms that modulate signalling may lead to the development of new therapies and may help to explain the mechanism of drug tolerance. PMID:9078236

  1. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

    PubMed Central

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-01-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs. PMID:26358189

  2. Methuselah/Methuselah-like G protein-coupled receptors constitute an ancient metazoan gene family

    PubMed Central

    de Mendoza, Alexandre; Jones, Jeffery W.; Friedrich, Markus

    2016-01-01

    Inconsistent conclusions have been drawn regarding the phylogenetic age of the Methuselah/Methuselah-like (Mth/Mthl) gene family of G protein-coupled receptors, the founding member of which regulates development and lifespan in Drosophila. Here we report the results from a targeted homolog search of 39 holozoan genomes and phylogenetic analysis of the conserved seven transmembrane domain. Our findings reveal that the Mth/Mthl gene family is ancient, has experienced numerous extinction and expansion events during metazoan evolution, and acquired the current definition of the Methuselah ectodomain during its exceptional expansion in arthropods. In addition, our findings identify Mthl1, Mthl5, Mthl14, and Mthl15 as the oldest Mth/Mthl gene family paralogs in Drosophila. Future studies of these genes have the potential to define ancestral functions of the Mth/Mthl gene family. PMID:26915348

  3. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination

    PubMed Central

    Yang, Hyun-Jeong; Vainshtein, Anna; Maik-Rachline, Galia; Peles, Elior

    2016-01-01

    While the formation of myelin by oligodendrocytes is critical for the function of the central nervous system, the molecular mechanism controlling oligodendrocyte differentiation remains largely unknown. Here we identify G protein-coupled receptor 37 (GPR37) as an inhibitor of late-stage oligodendrocyte differentiation and myelination. GPR37 is enriched in oligodendrocytes and its expression increases during their differentiation into myelin forming cells. Genetic deletion of Gpr37 does not affect the number of oligodendrocyte precursor cells, but results in precocious oligodendrocyte differentiation and hypermyelination. The inhibition of oligodendrocyte differentiation by GPR37 is mediated by suppression of an exchange protein activated by cAMP (EPAC)-dependent activation of Raf-MAPK-ERK1/2 module and nuclear translocation of ERK1/2. Our data suggest that GPR37 regulates central nervous system myelination by controlling the transition from early-differentiated to mature oligodendrocytes. PMID:26961174

  4. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination.

    PubMed

    Yang, Hyun-Jeong; Vainshtein, Anna; Maik-Rachline, Galia; Peles, Elior

    2016-01-01

    While the formation of myelin by oligodendrocytes is critical for the function of the central nervous system, the molecular mechanism controlling oligodendrocyte differentiation remains largely unknown. Here we identify G protein-coupled receptor 37 (GPR37) as an inhibitor of late-stage oligodendrocyte differentiation and myelination. GPR37 is enriched in oligodendrocytes and its expression increases during their differentiation into myelin forming cells. Genetic deletion of Gpr37 does not affect the number of oligodendrocyte precursor cells, but results in precocious oligodendrocyte differentiation and hypermyelination. The inhibition of oligodendrocyte differentiation by GPR37 is mediated by suppression of an exchange protein activated by cAMP (EPAC)-dependent activation of Raf-MAPK-ERK1/2 module and nuclear translocation of ERK1/2. Our data suggest that GPR37 regulates central nervous system myelination by controlling the transition from early-differentiated to mature oligodendrocytes.

  5. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. PMID:27284048

  6. In vitro expression and analysis of the 826 human G protein-coupled receptors.

    PubMed

    Lv, Xuechen; Liu, Junlin; Shi, Qiaoyun; Tan, Qiwen; Wu, Dong; Skinner, John J; Walker, Angela L; Zhao, Lixia; Gu, Xiangxiang; Chen, Na; Xue, Lu; Si, Pei; Zhang, Lu; Wang, Zeshi; Katritch, Vsevolod; Liu, Zhi-Jie; Stevens, Raymond C

    2016-05-01

    G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility. PMID:27085723

  7. G Protein-Coupled Receptor 87 (GPR87) Promotes Cell Proliferation in Human Bladder Cancer Cells.

    PubMed

    Zhang, Xia; Liu, Dage; Hayashida, Yushi; Okazoe, Homare; Hashimoto, Takeshi; Ueda, Nobufumi; Sugimoto, Mikio; Kakehi, Yoshiyuki

    2015-01-01

    G protein-coupled receptor 87 (GPR87) is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87) and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3), but not in the mutant p53 cells (HT1376 and J82). As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells. PMID:26473854

  8. The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

    PubMed Central

    Salon, John A.; Lodowski, David T.

    2011-01-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination. PMID:21969326

  9. ER-bound steps in the biosynthesis of G protein-coupled receptors.

    PubMed

    Nanoff, Christian; Freissmuth, Michael

    2012-01-01

    The polypeptide of a G protein-coupled receptor is inserted into the membrane of the endoplasmic reticulum while being translated and this process by itself may be sufficient to establish the proper receptor fold. X-ray structures reveal a common polypeptide topology with little variation in the alignment and orientation of the seven transmembrane segments, the proximal carboxyl terminus (C-tail) and parts of the extracellular loops. These define a structural core the stability of which probably represents a major criterion for the receptor to pass endoplasmic reticulum (ER) quality control; point mutations affecting the structure of the core have an extraordinary chance of causing receptor retention. In contrast, cytoplasmic loops 2 and 3 and the distal C-tail are poorly ordered at least in the absence of an interaction partner. Similarly, the amino terminal tail of rhodopsin-related receptors (but not of receptor subtypes where ligand binding requires a stable fold of the N-tail) is unlikely to establish a stable fold. These segments can cause ER retention when mutated to inappropriately expose hydrophobic peptide patches; to prevent protein aggregation chaperone molecules attach to them thus initiating selection for ER-associated degradation. It is less clear however if there are additional mechanisms to specifically survey the transmembrane core at the level of the lipid bilayer or if insufficient packing is detected due to misalignment of the cytoplasmic or extracellular face of the receptor. PMID:23161130

  10. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  11. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  12. Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

    PubMed Central

    Isom, Daniel G.; Dohlman, Henrik G.

    2015-01-01

    Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light- and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligand-binding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states. PMID:25902551

  13. Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay

    PubMed Central

    Yin, Hong; Chu, Alan; Li, Wei; Wang, Bin; Shelton, Fabiola; Otero, Francella; Nguyen, Deborah G.; Caldwell, Jeremy S.; Chen, Yu Alice

    2009-01-01

    A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the β-arrestin PathHunter™ assay system, a newly developed, generic GPCR assay format that measures β-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly “deorphaned” receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of ∼400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3′-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB2 receptor partial agonist, with binding affinity around 1 μm. The anti-inflammatory effect of 3,3′-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB2. PMID:19286662

  14. GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

    PubMed Central

    Zhang, Maoxiang; Davis, Jason E.; Li, Chunman; Gao, Jie; Huang, Wei; Lambert, Nevin A.; Terry, Alvin V.

    2016-01-01

    Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor. PMID:26811329

  15. Cloning of human genes encoding novel G protein-coupled receptors

    SciTech Connect

    Marchese, A.; Docherty, J.M.; Heiber, M.

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  16. Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

    PubMed

    Logez, Christel; Damian, Marjorie; Legros, Céline; Dupré, Clémence; Guéry, Mélody; Mary, Sophie; Wagner, Renaud; M'Kadmi, Céline; Nosjean, Olivier; Fould, Benjamin; Marie, Jacky; Fehrentz, Jean-Alain; Martinez, Jean; Ferry, Gilles; Boutin, Jean A; Banères, Jean-Louis

    2016-01-12

    G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment. PMID:26701065

  17. G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

    PubMed Central

    Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

    2012-01-01

    Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

  18. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

    PubMed

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-01-01

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

  19. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    SciTech Connect

    Pioszak, Augen A.; Xu, H. Eric

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  20. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions.

    PubMed

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala; Peisajovich, Sergio G

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease. PMID:27487915

  1. A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni.

    PubMed

    MacDonald, Kevin; Kimber, Michael J; Day, Tim A; Ribeiro, Paula

    2015-07-01

    The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor's basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR-like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting.

  2. Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies.

    PubMed

    Rich, Rebecca L; Miles, Adam R; Gale, Bruce K; Myszka, David G

    2009-03-01

    We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.

  3. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling

    PubMed Central

    Bock, Andreas; Merten, Nicole; Schrage, Ramona; Dallanoce, Clelia; Bätz, Julia; Klöckner, Jessica; Schmitz, Jens; Matera, Carlo; Simon, Katharina; Kebig, Anna; Peters, Lucas; Müller, Anke; Schrobang-Ley, Jasmin; Tränkle, Christian; Hoffmann, Carsten; De Amici, Marco; Holzgrabe, Ulrike; Kostenis, Evi; Mohr, Klaus

    2012-01-01

    Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling. PMID:22948826

  4. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists

    PubMed Central

    Cheng, Han; Lear-Rooney, Calli M.; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W.; Olinger, Gene G.

    2015-01-01

    ABSTRACT Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of

  5. Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

    PubMed

    Kahner, Bryan N; Quinton, Todd M; Langan, Sarah; Kunapuli, Satya P

    2006-09-01

    Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized 'secondary' receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Galphaq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors. PMID:16973501

  6. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  7. Novel screening assay for the selective detection of G-protein-coupled receptor heteromer signaling.

    PubMed

    van Rijn, Richard M; Harvey, Jessica H; Brissett, Daniela I; DeFriel, Julia N; Whistler, Jennifer L

    2013-01-01

    Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/μ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.

  8. Not lost in translation: Emerging clinical importance of the G protein-coupled estrogen receptor GPER.

    PubMed

    Barton, Matthias

    2016-07-01

    It has been 20years that the G protein-coupled estrogen receptor (GPER) was cloned as the orphan receptor GPR30 from multiple cellular sources, including vascular endothelial cells. Here, I will provide an overview of estrogen biology and the historical background leading to the discovery of rapid vascular estrogen signaling. I will also review the recent advances in the understanding of the mechanisms underlying GPER function, its role in physiology and disease, some of the currently available GPER-targeting drugs approved for clinical use such as SERMs (selective estrogen receptor modulators) and SERDs (selective estrogen receptor downregulators). Many of currently used drugs such as tamoxifen, raloxifene, or faslodex™/fulvestrant were discovered targeting GPER many years after they had been introduced to the clinics for entirely different purposes. This has important implications for the clinical use of these drugs and their modes of action, which I have termed 'reverse translational medicine'. In addition, environmental pollutants known as 'endocrine disruptors' have been found to bind to GPER. This article also discusses recent evidence in these areas as well as opportunities in translational clinical medicine and GPER research, including medical genetics, personalized medicine, prevention, and its theranostic use. PMID:26921679

  9. G Protein Coupled Receptors in Embryonic Stem Cells: A Role for Gs-Alpha Signaling

    PubMed Central

    Layden, Brian T.; Newman, Marsha; Chen, Fei; Fisher, Amanda; Lowe, William L.

    2010-01-01

    Background Identification of receptor mediated signaling pathways in embryonic stem (ES) cells is needed to facilitate strategies for cell replacement using ES cells. One large receptor family, largely uninvestigated in ES cells, is G protein coupled receptors (GPCRs). An important role for these receptors in embryonic development has been described, but little is known about GPCR expression in ES cells. Methodology/Principal Findings We have examined the expression profile of 343 different GPCRs in mouse ES cells demonstrating for the first time that a large number of GPCRs are expressed in undifferentiated and differentiating ES cells, and in many cases at high levels. To begin to define a role for GPCR signaling in ES cells, the impact of activating Gs-alpha, one of the major alpha subunits that couples to GPCRs, was investigated. Gs-alpha activation resulted in larger embryoid bodies (EBs), due, in part, to increased cell proliferation and prevented the time-related decline in expression of transcription factors important for maintaining ES cell pluripotency. Significance/Conclusions These studies suggest that Gs-alpha signaling contributes to ES cell proliferation and pluripotency and provide a framework for further investigation of GPCRs in ES cells. PMID:20161705

  10. Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa

    PubMed Central

    Li, Shizhong; Hauser, Frank; Skadborg, Signe K.; Nielsen, Stine V.; Kirketerp-Møller, Nikolaj; Grimmelikhuijzen, Cornelis J. P.

    2016-01-01

    Most multicellular animals belong to two evolutionary lineages, the Proto– and Deuterostomia, which diverged 640–760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily. PMID:27628442

  11. Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology.

    PubMed

    Dohlman, Henrik G

    2015-08-01

    Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human "GPCR-ome," including more than 150 uncharacterized "orphan" receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.

  12. Chemical biology methods for investigating G protein-coupled receptor signaling.

    PubMed

    Huber, Thomas; Sakmar, Thomas P

    2014-09-18

    G protein-coupled receptors (GPCRs) are targets for a quarter of prescription drugs. Despite recent progress in structural biology of GPCRs, only few key conformational states in the signal transduction process have been elucidated. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. Selective manipulation of individual steps in the GPCR activation scheme requires precise information about the kinetics of ligand binding and the dynamics of downstream signaling. One approach is to obtain time-resolved information using receptors tagged with fluorescent or structural probes. Recent advances allow for site-specific introduction of genetically encoded unnatural amino acids into expressed GPCRs. We describe how bioorthogonal functional groups on GPCRs enable the mapping of receptor-ligand interactions and how bioorthogonal chemical reactions can be used to introduce fluorescent labels for single-molecule fluorescence applications to study the kinetics and conformational dynamics of GPCR signaling complexes ("signalosomes").

  13. G-protein coupled receptor-mediated nutrient sensing and developmental control in Aspergillus nidulans.

    PubMed

    Brown, Neil Andrew; Dos Reis, Thaila Fernanda; Ries, Laure Nicolas Annick; Caldana, Camila; Mah, Jae-Hyung; Yu, Jae-Hyuk; Macdonald, Jeffrey M; Goldman, Gustavo Henrique

    2015-10-01

    Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G-protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre-formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient-sensing system functions upstream of the cAMP-PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans. PMID:26179439

  14. Fluorescent knock-in mice to decipher the physiopathological role of G protein-coupled receptors

    PubMed Central

    Ceredig, Rhian A.; Massotte, Dominique

    2015-01-01

    G protein-coupled receptors (GPCRs) modulate most physiological functions but are also critically involved in numerous pathological states. Approximately a third of marketed drugs target GPCRs, which places this family of receptors in the main arena of pharmacological pre-clinical and clinical research. The complexity of GPCR function demands comprehensive appraisal in native environment to collect in-depth knowledge of receptor physiopathological roles and assess the potential of therapeutic molecules. Identifying neurons expressing endogenous GPCRs is therefore essential to locate them within functional circuits whereas GPCR visualization with subcellular resolution is required to get insight into agonist-induced trafficking. Both remain frequently poorly investigated because direct visualization of endogenous receptors is often hampered by the lack of appropriate tools. Also, monitoring intracellular trafficking requires real-time visualization to gather in-depth knowledge. In this context, knock-in mice expressing a fluorescent protein or a fluorescent version of a GPCR under the control of the endogenous promoter not only help to decipher neuroanatomical circuits but also enable real-time monitoring with subcellular resolution thus providing invaluable information on their trafficking in response to a physiological or a pharmacological challenge. This review will present the animal models and discuss their contribution to the understanding of the physiopathological role of GPCRs. We will also address the drawbacks associated with this methodological approach and browse future directions. PMID:25610398

  15. G-Protein-Coupled Receptors: Next Generation Therapeutic Targets in Head and Neck Cancer?

    PubMed Central

    Kanazawa, Takeharu; Misawa, Kiyoshi; Misawa, Yuki; Uehara, Takayuki; Fukushima, Hirofumi; Kusaka, Gen; Maruta, Mikiko; Carey, Thomas E.

    2015-01-01

    Therapeutic outcome in head and neck squamous cell carcinoma (HNSCC) is poor in most advanced cases. To improve therapeutic efficiency, novel therapeutic targets and prognostic factors must be discovered. Our studies have identified several G protein-coupled receptors (GPCRs) as promising candidates. Significant epigenetic silencing of GPCR expression occurs in HNSCC compared with normal tissue, and is significantly correlated with clinical behavior. Together with the finding that GPCR activity can suppress tumor cell growth, this indicates that GPCR expression has potential utility as a prognostic factor. In this review, we discuss the roles that galanin receptor type 1 (GALR1) and type 2 (GALR2), tachykinin receptor type 1 (TACR1), and somatostatin receptor type 1 (SST1) play in HNSCC. GALR1 inhibits proliferation of HNSCC cells though ERK1/2-mediated effects on cell cycle control proteins such as p27, p57, and cyclin D1, whereas GALR2 inhibits cell proliferation and induces apoptosis in HNSCC cells. Hypermethylation of GALR1, GALR2, TACR1, and SST1 is associated with significantly reduced disease-free survival and a higher recurrence rate. Although their overall activities varies, each of these GPCRs has value as both a prognostic factor and a therapeutic target. These data indicate that further study of GPCRs is a promising strategy that will enrich pharmacogenomics and prognostic research in HNSCC. PMID:26251921

  16. G-Protein-Coupled Receptors: Next Generation Therapeutic Targets in Head and Neck Cancer?

    PubMed

    Kanazawa, Takeharu; Misawa, Kiyoshi; Misawa, Yuki; Uehara, Takayuki; Fukushima, Hirofumi; Kusaka, Gen; Maruta, Mikiko; Carey, Thomas E

    2015-08-01

    Therapeutic outcome in head and neck squamous cell carcinoma (HNSCC) is poor in most advanced cases. To improve therapeutic efficiency, novel therapeutic targets and prognostic factors must be discovered. Our studies have identified several G protein-coupled receptors (GPCRs) as promising candidates. Significant epigenetic silencing of GPCR expression occurs in HNSCC compared with normal tissue, and is significantly correlated with clinical behavior. Together with the finding that GPCR activity can suppress tumor cell growth, this indicates that GPCR expression has potential utility as a prognostic factor. In this review, we discuss the roles that galanin receptor type 1 (GALR1) and type 2 (GALR2), tachykinin receptor type 1 (TACR1), and somatostatin receptor type 1 (SST1) play in HNSCC. GALR1 inhibits proliferation of HNSCC cells though ERK1/2-mediated effects on cell cycle control proteins such as p27, p57, and cyclin D1, whereas GALR2 inhibits cell proliferation and induces apoptosis in HNSCC cells. Hypermethylation of GALR1, GALR2, TACR1, and SST1 is associated with significantly reduced disease-free survival and a higher recurrence rate. Although their overall activities varies, each of these GPCRs has value as both a prognostic factor and a therapeutic target. These data indicate that further study of GPCRs is a promising strategy that will enrich pharmacogenomics and prognostic research in HNSCC.

  17. Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa.

    PubMed

    Li, Shizhong; Hauser, Frank; Skadborg, Signe K; Nielsen, Stine V; Kirketerp-Møller, Nikolaj; Grimmelikhuijzen, Cornelis J P

    2016-01-01

    Most multicellular animals belong to two evolutionary lineages, the Proto- and Deuterostomia, which diverged 640-760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily. PMID:27628442

  18. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    SciTech Connect

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  19. Distinct G protein-coupled receptor recycling pathways allow spatial control of downstream G protein signaling.

    PubMed

    Bowman, Shanna Lynn; Shiwarski, Daniel John; Puthenveedu, Manojkumar A

    2016-09-26

    G protein-coupled receptors (GPCRs) are recycled via a sequence-dependent pathway that is spatially and biochemically distinct from bulk recycling. Why there are two distinct recycling pathways from the endosome is a fundamental question in cell biology. In this study, we show that the separation of these two pathways is essential for normal spatial encoding of GPCR signaling. The prototypical β-2 adrenergic receptor (B2AR) activates Gα stimulatory protein (Gαs) on the endosome exclusively in sequence-dependent recycling tubules marked by actin/sorting nexin/retromer tubular (ASRT) microdomains. B2AR was detected in an active conformation in bulk recycling tubules, but was unable to activate Gαs. Protein kinase A phosphorylation of B2AR increases the fraction of receptors localized to ASRT domains and biases the downstream transcriptional effects of B2AR to genes controlled by endosomal signals. Our results identify the physiological relevance of separating GPCR recycling from bulk recycling and suggest a mechanism to tune downstream responses of GPCR signaling by manipulating the spatial origin of G protein signaling. PMID:27646272

  20. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    PubMed

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles.

  1. The structural evolution of a P2Y-like G-protein-coupled receptor.

    PubMed

    Schulz, Angela; Schöneberg, Torsten

    2003-09-12

    Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution approximately 43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains. PMID:12835326

  2. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  3. A crystal clear solution for determining G-protein-coupled receptor structures.

    PubMed

    Tate, Christopher G

    2012-09-01

    G-protein-coupled receptors (GPCRs) are medically important membrane proteins that are targeted by over 30% of small molecule drugs. At the time of writing, 15 unique GPCR structures have been determined, with 77 structures deposited in the PDB database, which offers new opportunities for drug development and for understanding the molecular mechanisms of GPCR activation. Many different factors have contributed to this success, but if there is one single factor that can be singled out as the foundation for producing well-diffracting GPCR crystals, it is the stabilisation of the detergent-solubilised receptor-ligand complex. This review will focus predominantly on one of the successful strategies for the stabilisation of GPCRs, namely the thermostabilisation of GPCRs using systematic mutagenesis coupled with thermostability assays. Structures of thermostabilised GPCRs bound to a wide variety of ligands have been determined, which has led to an understanding of ligand specificity; why some ligands act as agonists as opposed to partial or inverse agonists; and the structural basis for receptor activation.

  4. Distinct G protein-coupled receptor recycling pathways allow spatial control of downstream G protein signaling.

    PubMed

    Bowman, Shanna Lynn; Shiwarski, Daniel John; Puthenveedu, Manojkumar A

    2016-09-26

    G protein-coupled receptors (GPCRs) are recycled via a sequence-dependent pathway that is spatially and biochemically distinct from bulk recycling. Why there are two distinct recycling pathways from the endosome is a fundamental question in cell biology. In this study, we show that the separation of these two pathways is essential for normal spatial encoding of GPCR signaling. The prototypical β-2 adrenergic receptor (B2AR) activates Gα stimulatory protein (Gαs) on the endosome exclusively in sequence-dependent recycling tubules marked by actin/sorting nexin/retromer tubular (ASRT) microdomains. B2AR was detected in an active conformation in bulk recycling tubules, but was unable to activate Gαs. Protein kinase A phosphorylation of B2AR increases the fraction of receptors localized to ASRT domains and biases the downstream transcriptional effects of B2AR to genes controlled by endosomal signals. Our results identify the physiological relevance of separating GPCR recycling from bulk recycling and suggest a mechanism to tune downstream responses of GPCR signaling by manipulating the spatial origin of G protein signaling.

  5. The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components.

    PubMed

    Borroto-Escuela, Dasiel O; Brito, Ismel; Romero-Fernandez, Wilber; Di Palma, Michael; Oflijan, Julia; Skieterska, Kamila; Duchou, Jolien; Van Craenenbroeck, Kathleen; Suárez-Boomgaard, Diana; Rivera, Alicia; Guidolin, Diego; Agnati, Luigi F; Fuxe, Kjell

    2014-05-14

    G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

  6. Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors.

    PubMed

    Jean-Charles, P-Y; Snyder, J C; Shenoy, S K

    2016-01-01

    The seven-transmembrane containing G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. Transmembrane signaling by GPCRs is fundamental to many aspects of physiology including vision, olfaction, cardiovascular, and reproductive functions as well as pain, behavior and psychomotor responses. The duration and magnitude of signal transduction is tightly controlled by a series of coordinated trafficking events that regulate the cell-surface expression of GPCRs at the plasma membrane. Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs. Of the various molecular mechanisms that impart selectivity, sensitivity and strength of transmembrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR. Ubiquitination of proteins was originally discovered in the context of lysosome-independent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes. In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling. To date about 40 GPCRs are known to be ubiquitinated. For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes. This chapter will focus on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

  7. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  8. The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components.

    PubMed

    Borroto-Escuela, Dasiel O; Brito, Ismel; Romero-Fernandez, Wilber; Di Palma, Michael; Oflijan, Julia; Skieterska, Kamila; Duchou, Jolien; Van Craenenbroeck, Kathleen; Suárez-Boomgaard, Diana; Rivera, Alicia; Guidolin, Diego; Agnati, Luigi F; Fuxe, Kjell

    2014-01-01

    G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html. PMID:24830558

  9. Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling.

    PubMed

    Marchese, Adriano; Trejo, Joann

    2013-03-01

    G protein-coupled receptors (GPCRs) belong to one of the largest family of signaling receptors in the mammalian genome [1]. GPCRs elicit cellular responses to multiple diverse stimuli and play essential roles in human health and disease. GPCRs have important clinical implications in various diseases and are the targets of approximately 25-50% of all marketed drugs [2,3]. Understanding how GPCRs are regulated is essential to delineating their role in normal physiology and in the pathophysiology of several diseases. Given the vast number and diversity of GPCRs, it is likely that multiple mechanisms exist to regulate GPCR function. While GPCR signaling is typically regulated by desensitization and endocytosis mediated by phosphorylation and β-arrestins, it can also be modulated by ubiquitination. Ubiquitination is emerging an important regulatory process that may have unique roles in governing GPCR trafficking and signaling. Recent studies have revealed a mechanistic link between GPCR phosphorylation, β-arrestins and ubiquitination that may be applicable to some GPCRs but not others. While the function of ubiquitination is generally thought to promote receptor endocytosis and endosomal sorting, recent studies have revealed that ubiquitination also plays an important role in positive regulation of GPCR signaling. Here, we will review recent developments in our understanding of how ubiquitin regulates GPCR endocytic trafficking and how it contributes to signal transduction induced by GPCR activation.

  10. TLR4 Signaling augments monocyte chemotaxis by regulating G protein-coupled receptor kinase 2 translocation.

    PubMed

    Liu, Zheng; Jiang, Yong; Li, Yuehua; Wang, Juan; Fan, Liyan; Scott, Melanie J; Xiao, Guozhi; Li, Song; Billiar, Timothy R; Wilson, Mark A; Fan, Jie

    2013-07-15

    Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein-coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphorylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS-TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1-induced monocyte migration. We also show that LPS, through p38 MAPK signaling, induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.

  11. G protein-coupled receptor 35: an emerging target in inflammatory and cardiovascular disease

    PubMed Central

    Divorty, Nina; Mackenzie, Amanda E.; Nicklin, Stuart A.; Milligan, Graeme

    2015-01-01

    G protein-coupled receptor 35 (GPR35) is an orphan receptor, discovered in 1998, that has garnered interest as a potential therapeutic target through its association with a range of diseases. However, a lack of pharmacological tools and the absence of convincingly defined endogenous ligands have hampered the understanding of function necessary to exploit it therapeutically. Although several endogenous molecules can activate GPR35 none has yet been confirmed as the key endogenous ligand due to reasons that include lack of biological specificity, non-physiologically relevant potency and species ortholog selectivity. Recent advances have identified several highly potent synthetic agonists and antagonists, as well as agonists with equivalent potency at rodent and human orthologs, which will be useful as tool compounds. Homology modeling and mutagenesis studies have provided insight into the mode of ligand binding and possible reasons for the species selectivity of some ligands. Advances have also been made in determining the role of the receptor in disease. In the past, genome-wide association studies have associated GPR35 with diseases such as inflammatory bowel disease, type 2 diabetes, and coronary artery disease. More recent functional studies have implicated it in processes as diverse as heart failure and hypoxia, inflammation, pain transduction and synaptic transmission. In this review, we summarize the progress made in understanding the molecular pharmacology, downstream signaling and physiological function of GPR35, and discuss its emerging potential applications as a therapeutic target. PMID:25805994

  12. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    PubMed Central

    Di Roberto, Raphaël B.; Chang, Belinda; Trusina, Ala; Peisajovich, Sergio G.

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor–ligand pair can evolve through network-altering mutations independently of receptor–ligand binding, and suggest a potential role for such mutations in disease. PMID:27487915

  13. Regulation of the Hippo-YAP pathway by G-protein coupled receptor signaling

    PubMed Central

    Yu, Fa-Xing; Zhao, Bin; Panupinthu, Nattapon; Jewell, Jenna L.; Lian, Ian; Wang, Lloyd H.; Zhao, Jiagang; Yuan, Haixin; Tumaneng, Karen; Li, Hairi; Fu, Xiang-Dong; Mills, Gordon B.; Guan, Kun-Liang

    2012-01-01

    SUMMARY The Hippo pathway is crucial in organ size control and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here we report that the Hippo pathway is regulated by G-protein coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2 thereby activating YAP and TAZ transcription co-activators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G-protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR. PMID:22863277

  14. Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55.

    PubMed

    Bouskila, Joseph; Harrar, Vanessa; Javadi, Pasha; Casanova, Christian; Hirabayashi, Yoshio; Matsuo, Ichiro; Ohyama, Jyunpei; Bouchard, Jean-François; Ptito, Maurice

    2016-01-01

    The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the monkey retina, suggesting its possible role in scotopic vision. To test this hypothesis, we recorded full-field electroretinograms (ERGs) after the intravitreal injection of the GPR55 agonist lysophosphatidylglucoside (LPG) or the selective GPR55 antagonist CID16020046 (CID), under light- and dark-adapted conditions. Thirteen vervet monkeys (Chlorocebus sabaeus) were used in this study: four controls (injected with the vehicle dimethyl sulfoxide, DMSO), four injected with LPG and five with CID. We analyzed amplitudes and latencies of the a-wave (photoreceptor responses) and the b-wave (rod and cone system responses) of the ERG. Our results showed that after injection of LPG, the amplitude of the scotopic b-wave was significantly higher, whereas after the injection of CID, it was significantly decreased, compared to the vehicle (DMSO). On the other hand, the a-wave amplitude, and the a-wave and b-wave latencies, of the scotopic ERG responses were not significantly affected by the injection of either compound. Furthermore, the photopic ERG waveforms were not affected by either drug. These results support the hypothesis that GPR55 plays an instrumental role in mediating scotopic vision. PMID:27485069

  15. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.

    PubMed

    Singh, Nidhi; Yadav, Manisha; Singh, Abhishek Kumar; Kumar, Harish; Dwivedi, Shailendra Kumar Dhar; Mishra, Jay Sharan; Gurjar, Anagha; Manhas, Amit; Chandra, Sharat; Yadav, Prem Narayan; Jagavelu, Kumaravelu; Siddiqi, Mohammad Imran; Trivedi, Arun Kumar; Chattopadhyay, Naibedya; Sanyal, Sabyasachi

    2014-05-01

    The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.

  16. The roles played by highly truncated splice variants of G protein-coupled receptors

    PubMed Central

    2012-01-01

    Alternative splicing of G protein-coupled receptor (GPCR) genes greatly increases the total number of receptor isoforms which may be expressed in a cell-dependent and time-dependent manner. This increased diversity of cell signaling options caused by the generation of splice variants is further enhanced by receptor dimerization. When alternative splicing generates highly truncated GPCRs with less than seven transmembrane (TM) domains, the predominant effect in vitro is that of a dominant-negative mutation associated with the retention of the wild-type receptor in the endoplasmic reticulum (ER). For constitutively active (agonist-independent) GPCRs, their attenuated expression on the cell surface, and consequent decreased basal activity due to the dominant-negative effect of truncated splice variants, has pathological consequences. Truncated splice variants may conversely offer protection from disease when expression of co-receptors for binding of infectious agents to cells is attenuated due to ER retention of the wild-type co-receptor. In this review, we will see that GPCRs retained in the ER can still be functionally active but also that highly truncated GPCRs may also be functionally active. Although rare, some truncated splice variants still bind ligand and activate cell signaling responses. More importantly, by forming heterodimers with full-length GPCRs, some truncated splice variants also provide opportunities to generate receptor complexes with unique pharmacological properties. So, instead of assuming that highly truncated GPCRs are associated with faulty transcription processes, it is time to reassess their potential benefit to the host organism. PMID:22938630

  17. Biased agonism at G protein-coupled receptors: the promise and the challenges--a medicinal chemistry perspective.

    PubMed

    Shonberg, Jeremy; Lopez, Laura; Scammells, Peter J; Christopoulos, Arthur; Capuano, Ben; Lane, J Robert

    2014-11-01

    Historically, determination of G protein-coupled receptor (GPCR) ligand efficacy has often been restricted to identifying the ligand as an agonist or antagonist at a given signaling pathway. This classification was deemed sufficient to predict compound efficacy at all signaling endpoints, including the therapeutically relevant one(s). However, it is now apparent that ligands acting at the same GPCR can stabilize multiple, distinct, receptor conformations linked to different functional outcomes. This phenomenon, known as biased agonism, stimulus bias, or functional selectivity offers the opportunity to separate on-target therapeutic effects from side effects through the design of drugs that show pathway selectivity. However, the medicinal chemist faces numerous challenges to develop biased ligands, including the detection and quantification of biased agonism. This review summarizes the current state of the field of research into biased agonism at GPCRs, with a particular focus on efforts to relate biased agonism to ligand structure.

  18. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    PubMed

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling. PMID:26928539

  19. Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin

    PubMed Central

    Reyes-Alcaraz, Arfaxad; Martínez-Archundia, Marlet; Ramon, Eva; Garriga, Pere

    2011-01-01

    Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable. PMID:22261069

  20. Genomic and supragenomic structure of the nucleotide-like G-protein-coupled receptor GPR34.

    PubMed

    Engemaier, Eva; Römpler, Holger; Schöneberg, Torsten; Schulz, Angela

    2006-02-01

    Directed cloning approaches and large-scale sequencing of several vertebrate genomes unveiled many new members of the G-protein-coupled receptor (GPCR) superfamily, among them GPR34. Initial studies showed that GPR34 is an evolutionarily old GPCR structurally related to a group of ADP-like receptors. To gain insight into the genomic organization, regulation of expression, and supragenomic diversification of GPR34 several vertebrate species were analyzed. In contrast to the obviously intronless coding region GPR34 displays an evolutionary preserved 5' noncoding intron-exon structure. Further, an alternatively used cryptic intron was identified within the coding region, which shortens the N terminus by 47 amino acids. Ubiquitous expression of GPR34 is driven by genomic sequences upstream of at least two transcriptional start regions in mouse and rat but only one region in human. In rodents, both promoters are active in all tissues investigated, but the level of activity is tissue-specific. At the translational level, several conserved in-frame AUGs within the first 150 bp of the coding region may serve as start points for translation in human and other mammals. Combinatory mutagenesis and expression of reporter constructs confirmed these multiple translational start points and revealed a preference for the second in-frame AUG in human GPR34. Our data show that multiple translation initiation starts and alternative splicing contribute to the supragenomic diversification of GPR34. PMID:16338117

  1. Structural mechanism of G protein activation by G protein-coupled receptor.

    PubMed

    Duc, Nguyen Minh; Kim, Hee Ryung; Chung, Ka Young

    2015-09-15

    G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate physiology and pathology of various organs. Consequently, about 40% of drugs in the market targets GPCRs. Heterotrimeric G proteins are composed of α, β, and γ subunits, and act as the key downstream signaling molecules of GPCRs. The structural mechanism of G protein activation by GPCRs has been of a great interest, and a number of biochemical and biophysical studies have been performed since the late 80's. These studies investigated the interface between GPCR and G proteins and the structural mechanism of GPCR-induced G protein activation. Recently, arrestins are also reported to be important molecular switches in GPCR-mediated signal transduction, and the physiological output of arrestin-mediated signal transduction is different from that of G protein-mediated signal transduction. Understanding the structural mechanism of the activation of G proteins and arrestins would provide fundamental information for the downstream signaling-selective GPCR-targeting drug development. This review will discuss the structural mechanism of GPCR-induced G protein activation by comparing previous biochemical and biophysical studies.

  2. G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

    PubMed

    Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

    2015-01-01

    Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer.

  3. A chemical screen identifies class A G-protein coupled receptors as regulators of cilia

    PubMed Central

    Avasthi, Prachee; Marley, Aaron; Lin, Henry; Gregori-Puigjane, Elisabet; Shoichet, Brian K.; von Zastrow, Mark; Marshall, Wallace F.

    2012-01-01

    Normal cilia length and motility are critical for proper cellular function. Prior studies of the regulation of ciliary structure and length have primarily focused on the intraflagellar transport machinery and motor proteins required for ciliary assembly and disassembly. However, several mutants with abnormal length flagella highlight the importance of signaling proteins as well. In this study, an unbiased chemical screen was performed to uncover signaling pathways that are critical for ciliogenesis and length regulation using flagella of the green alga Chlamydomonas reinhardtii as a model. The annotated Sigma LOPAC1280 chemical library was screened for effects on flagellar length, motility and severing as well as cell viability. Assay data were clustered to identify pathways regulating flagella. The most frequently target found to be involved in flagellar length regulation was the family of dopamine binding G-protein coupled receptors (GPCRs). In mammalian cells, cilium length could indeed be altered with expression of the dopamine D1 receptor. Our screen thus reveals signaling pathways that are potentially critical for ciliary formation, resorption, and length maintenance, which represent candidate targets for therapeutic intervention of disorders involving ciliary malformation and malfunction. PMID:22375814

  4. Functional modulation of G-protein coupled receptors during Parkinson disease-like neurodegeneration.

    PubMed

    Jenkins, Bruce G; Zhu, Aijun; Poutiainen, Pekka; Choi, Ji-Kyung; Kil, Kun-Eek; Zhang, Zhaoda; Kuruppu, Darshini; Aytan, Nurgul; Dedeoglu, Alpaslan; Brownell, Anna-Liisa

    2016-09-01

    G-protein coupled dopamine and metabotropic glutamate receptors (mGlu) can modulate neurotransmission during Parkinson's disease (PD)-like neurodegeneration. PET imaging studies in a unilateral dopamine denervation model (6-OHDA) showed a significant inverse correlation of presynaptic mGlu4 and postsynaptic mGlu5 expression in the striatum and rapidly declining mGlu4 and enhanced mGlu5 expression in the hippocampus during progressive degeneration over time. Immunohistochemical studies verified the decreased mGlu4 expression in the hippocampus on the lesion side but did not show difference in mGlu5 expression between lesion and control side. Pharmacological MRI studies showed enhanced hemodynamic response in several brain areas on the lesion side compared to the control side after challenge with mGlu4 positive allosteric modulator or mGlu5 negative allosteric modulator. However, mGlu4 response was biphasic having short enhancement followed by negative response on both sides of brain. Studies in mGlu4 expressing cells demonstrated that glutamate induces cooperative increase in binding of mGlu4 ligands - especially at high glutamate levels consistent with in vivo concentration. This suggests that mGlu allosteric modulators as drug candidates will be highly sensitive to changes in glutamate concentration and hence metabolic state. These experiments demonstrate the importance of the longitudinal imaging studies to investigate temporal changes in receptor functions to obtain individual response for experimental drugs. PMID:26581500

  5. Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

    PubMed

    Tran, Cawa; Hadfield, Michael G

    2012-04-01

    Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae. PMID:22589403

  6. Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

    PubMed Central

    Rahmeh, Rita; Damian, Marjorie; Cottet, Martin; Orcel, Hélène; Mendre, Christiane; Durroux, Thierry; Sharma, K. Shivaji; Durand, Grégory; Pucci, Bernard; Trinquet, Eric; Zwier, Jurriaan M.; Deupi, Xavier; Bron, Patrick; Banères, Jean-Louis; Mouillac, Bernard; Granier, Sébastien

    2012-01-01

    G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs. PMID:22493271

  7. Allosteric inhibition of g-protein coupled receptor oligomerization: strategies and challenges for drug development.

    PubMed

    Hurevich, Mattan; Talhami, Alaa; Shalev, Deborah E; Gilon, Chaim

    2014-01-01

    G-protein coupled receptors (GPCRs) mediate a large number of biological pathways and are major therapeutic targets. One of the most exiting phenomena of GPCRs is their ability to interact with other GPCRs. GPCRGPCR interactions, also known as GPCR oligomerization, may create various functional entities such as homo- and heterodimers and also form complex multimeric GPCR clusters. In many biological systems, GPCR-GPCR interactions are crucial for signal regulation. The interaction with other receptors results in allosteric modifications of GPCRs through conformational changes. Allosteric inhibition of GPCRs is considered an attractive strategy for drug development and does not involve targeting the orthosteric site. Understanding the nature of GPCR-GPCR interactions is mandatory for developing allosteric inhibitors. Studying GPCR-GPCR interactions is a challenging task and many methods have been developed to analyze these events. This review will highlight some of the methods developed to study GPCR-GPCR interactions and will describe pivotal studies that provided the basic understanding of the importance of GPCR oligomerization. We will also describe the significance of GPCR interaction networks for drug development. Recent studies will be reviewed to illustrate the use of state-of-the-art biophysical and spectroscopic methods for the discovery of GPCR oligomerization modulators.

  8. Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression

    PubMed Central

    Lu, Min; Staszewski, Lena; Echeverri, Fernando; Xu, Hong; Moyer, Bryan D

    2004-01-01

    Background Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells. Results To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway. Conclusions These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination. PMID:15369603

  9. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    PubMed Central

    Culhane, Kelly J.; Liu, Yuting; Cai, Yingying; Yan, Elsa C. Y.

    2015-01-01

    Although family B G protein-coupled receptors (GPCRs) contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs. PMID:26594176

  10. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    PubMed

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling.

  11. G Protein-Coupled Estrogen Receptor in Energy Homeostasis and Obesity Pathogenesis

    PubMed Central

    Shi, Haifei; Dharshan Senthil Kumar, Shiva Priya; Liu, Xian

    2013-01-01

    Obesity and its related metabolic diseases have reached a pandemic level worldwide. There are sex differences in the prevalence of obesity and its related metabolic diseases, with men being more vulnerable than women; however, the prevalence of these disorders increases dramatically in women after menopause, suggesting that sex steroid hormone estrogens play key protective roles against development of obesity and metabolic diseases. Estrogens are important regulators of several aspects of metabolism, including body weight and body fat, caloric intake and energy expenditure, and glucose and lipid metabolism in both males and females. Estrogens act in complex ways on their nuclear estrogen receptors (ERs) ERα and ERβ and transmembrane ERs such as G protein-coupled estrogen receptor. Genetic tools, such as different lines of knockout mouse models, and pharmacological agents, such as selective agonists and antagonists, are available to study function and signaling mechanisms of ERs. We provide an overview of the evidence for the physiological and cellular actions of ERs in estrogen-dependent processes in the context of energy homeostasis and body fat regulation and discuss its pathology that leads to obesity and related metabolic states. PMID:23317786

  12. The Evolving Impact of G Protein-Coupled Receptor Kinases in Cardiac Health and Disease

    PubMed Central

    Sato, Priscila Y.; Chuprun, J. Kurt; Schwartz, Mathew; Koch, Walter J.

    2015-01-01

    G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure. PMID:25834229

  13. G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy

    PubMed Central

    White, James P.; Wrann, Christiane D.; Rao, Rajesh R.; Nair, Sreekumaran K.; Jedrychowski, Mark P.; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P.; Ruas, Jorge L.; Hornberger, Troy A.; Wu, Zhidan; Glass, David J.; Piao, Xianhua; Spiegelman, Bruce M.

    2014-01-01

    Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4–induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise. PMID:25336758

  14. Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

    PubMed Central

    Stoveken, Hannah M.; Hajduczok, Alexander G.; Xu, Lei; Tall, Gregory G.

    2015-01-01

    The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease. PMID:25918380

  15. Global Survey of Canonical Aspergillus flavus G Protein-Coupled Receptors

    PubMed Central

    Affeldt, Katharyn J.; Carrig, Joseph; Amare, Meareg

    2014-01-01

    ABSTRACT G protein-coupled receptors (GPCRs) are transmembrane receptors that relay signals from the external environment inside the cell, allowing an organism to adapt to its surroundings. They are known to detect a vast array of ligands, including sugars, amino acids, pheromone peptides, nitrogen sources, oxylipins, and light. Despite their prevalence in fungal genomes, very little is known about the functions of filamentous fungal GPCRs. Here we present the first full-genome assessment of fungal GPCRs through characterization of null mutants of all 15 GPCRs encoded by the aflatoxin-producing fungus Aspergillus flavus. All strains were assessed for growth, development, ability to produce aflatoxin, and response to carbon sources, nitrogen sources, stress agents, and lipids. Most GPCR mutants were aberrant in one or more response processes, possibly indicative of cross talk in downstream signaling pathways. Interestingly, the biological defects of the mutants did not correspond with assignment to established GPCR classes; this is likely due to the paucity of data for characterized fungal GPCRs. Many of the GPCR transcripts were differentially regulated under various conditions as well. The data presented here provide an extensive overview of the full set of GPCRs encoded by A. flavus and provide a framework for analysis in other fungal species. PMID:25316696

  16. GPCR-ModSim: A comprehensive web based solution for modeling G-protein coupled receptors

    PubMed Central

    Esguerra, Mauricio; Siretskiy, Alexey; Bello, Xabier; Sallander, Jessica; Gutiérrez-de-Terán, Hugo

    2016-01-01

    GPCR-ModSim (http://open.gpcr-modsim.org) is a centralized and easy to use service dedicated to the structural modeling of G-protein Coupled Receptors (GPCRs). 3D molecular models can be generated from amino acid sequence by homology-modeling techniques, considering different receptor conformations. GPCR-ModSim includes a membrane insertion and molecular dynamics (MD) equilibration protocol, which can be used to refine the generated model or any GPCR structure uploaded to the server, including if desired non-protein elements such as orthosteric or allosteric ligands, structural waters or ions. We herein revise the main characteristics of GPCR-ModSim and present new functionalities. The templates used for homology modeling have been updated considering the latest structural data, with separate profile structural alignments built for inactive, partially-active and active groups of templates. We have also added the possibility to perform multiple-template homology modeling in a unique and flexible way. Finally, our new MD protocol considers a series of distance restraints derived from a recently identified conserved network of helical contacts, allowing for a smoother refinement of the generated models which is particularly advised when there is low homology to the available templates. GPCR- ModSim has been tested on the GPCR Dock 2013 competition with satisfactory results. PMID:27166369

  17. Conformational Dynamics of Single G Protein-Coupled Receptors in Solution

    PubMed Central

    Bockenhauer, Samuel; Fürstenberg, Alexandre; Yao, Xiao Jie; Kobilka, Brian K.; Moerner, W. E.

    2011-01-01

    G Protein-Coupled Receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β2-Adrenergic Receptors (β2ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently-labeled β2AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β2ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with stretching exponent β ~ 0.5, suggesting protein dynamics over a range of timescales. PMID:21928818

  18. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    PubMed

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

  19. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

    PubMed Central

    Martin, Adam L.; Steurer, Michael A.; Aronstam, Robert S.

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

  20. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    PubMed

    Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

  1. Chemical modification of Class II G-protein coupled receptor ligands

    PubMed Central

    Chapter, Megan C.; White, Caitlin M.; De Ridder, Angela; Chadwick, Wayne; Martin, Bronwen; Maudsley, Stuart

    2009-01-01

    Recent research and clinical data have begun to demonstrate the huge potential therapeutic importance of ligands that modulate the activity of the secretin-like, Class II, G-protein coupled receptors (GPCRs). Ligands that can modulate the activity of these Class II GPCRs may have important clinical roles in the treatment of a wide variety of conditions such as osteoporosis, diabetes, amyotrophic lateral sclerosis and autism spectrum disorders. While these receptors present important new therapeutic targets, the large glycoprotein nature of their cognate ligands poses many problems with respect to therapeutic peptidergic drug design. These native peptides often exhibit poor bioavailability, metabolic instability, poor receptor selectivity and resultant low potencies in vivo. Recently, increased attention has been paid to the structural modification of these peptides to enhance their therapeutic efficacy. Successful modification strategies have included D-amino acid substitutions, selective truncation, and fatty acid acylation of the peptide. Through these and other processes, these novel peptide ligand analogs can demonstrate enhanced receptor subtype selectivity, directed signal transduction pathway activation, resistance to proteolytic degradation, and improved systemic bioavailability. In the future, it is likely, through additional modification strategies such as addition of circulation-stabilizing transferrin moieties, that the therapeutic pharmacopeia of drugs targeted towards Class II secretin-like receptors may rival that of the Class I rhodopsin-like receptors that currently provide the majority of clinically used GPCR-based therapeutics. Currently, Class II-based drugs include synthesized analogues of vasoactive intestinal peptide for type 2 diabetes or parathyroid hormone for osteoporosis. PMID:19686775

  2. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  3. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  4. Consequences of splice variation on Secretin family G protein-coupled receptor function

    PubMed Central

    Furness, Sebastian GB; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick M

    2012-01-01

    The Secretin family of GPCRs are endocrine peptide hormone receptors that share a common genomic organization and are the subject of a wide variety of alternative splicing. All GPCRs contain a central seven transmembrane domain responsible for transducing signals from the outside of the cell as well as extracellular amino and intracellular carboxyl termini. Members of the Secretin receptor family have a relatively large N-terminus and a variety of lines of evidence support a common mode of ligand binding and a common ligand binding fold. These receptors are best characterized as coupling to intracellular signalling pathways via Gαs and Gαq but are also reported to couple to a multitude of other signalling pathways. The intracellular loops are implicated in regulating the interaction between the receptor and heterotrimeric G protein complexes. Alternative splicing of exons encoding both the extracellular N-terminal domain as well as the extracellular loops of some family members has been reported and as expected these splice variants display altered ligand affinity as well as differential activation by endogenous ligands. Various forms of alternative splicing have also been reported to alter intracellular loops 1 and 3 as well as the C-terminus and as one might expect these display differences in signalling bias towards downstream effectors. These diverse pharmacologies require that the physiological role of these splice variants be addressed but should provide unique opportunities for drug design and development. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1 PMID:21718310

  5. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction

    PubMed Central

    Bradley, Sophie J.; Iglesias, Max Maza; Kong, Kok Choi; Butcher, Adrian J.; Plouffe, Bianca; Goupil, Eugénie; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; LeGouill, Christian; Russell, Kirsty; Laporte, Stéphane A.; König, Gabriele M.; Kostenis, Evi; Bouvier, Michel; Chung, Kian Fan; Amrani, Yassine; Tobin, Andrew B.

    2016-01-01

    G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR–biased ligands with important implications for drug discovery. PMID:27071102

  6. Feeding and the rhodopsin family g-protein coupled receptors in nematodes and arthropods.

    PubMed

    Cardoso, João C R; Félix, Rute C; Fonseca, Vera G; Power, Deborah M

    2012-01-01

    In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologs of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans), and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologs of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors. PMID:23264768

  7. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  8. Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107.

    PubMed

    Yosten, Gina L C; Redlinger, Lauren J; Samson, Willis K

    2012-11-01

    Neuronostatin, derived from the somatostatin preprohormone, is a recently described peptide that is produced by several tissues involved in cardiovascular regulation and metabolism, including the hypothalamus. Injection of neuronostatin into the lateral cerebroventricle led to a dose-related increase in mean arterial pressure (MAP) in rats. Any attempt to inhibit the production of neuronostatin would alter somatostatin production as well, making determination of the physiological relevance of the peptide's pharmacologic effects by compromise of production approaches impossible. Therefore, we employed an alternative approach to identify and compromise the production of the neuronostatin receptor. Because neuronostatin was shown to signal via a PKA-dependent mechanism, we hypothesized that the neuronostatin receptor was a G protein-coupled receptor (GPCR), in particular, one of the orphan GPCRs for which the ligand is unknown. Therefore, we screened neuronostatin-responsive tissues, including hypothalamus, heart, pancreatic α-cells, and the gastric tumor cell line KATOIII, for expression of orphan GPCRs. Four orphan GPCRs were expressed by all cell types, including GPR56 and GPR107. Knockdown of GPR107, but not GPR56 or GPR146, led to a loss of responsiveness to neuronostatin by KATOIII cells. Rats injected with siRNA directed against GPR107 (2 μg/day for 2 days) into the lateral cerebroventricle did not exhibit an increase in MAP in response to neuronostatin treatment. Rats with compromised GPR107 expression also displayed blunted reactivity in a baroreflex sensitivity test, indicating that GPR107 and neuronostatin may be important regulators of cardiovascular function. Thus, GPR107 is a promising candidate receptor for neuronostatin, and neuronostatin, interacting with GPR107, may play an important role in the central control of cardiovascular function. PMID:22933024

  9. Self-organized criticality in proteins: Hydropathic roughening profiles of G-protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2013-03-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein conformational functionality is often dominated by long-range hydrophobic or hydrophilic interactions which both drive protein compaction and mediate protein-protein interactions. Superfamily transmembrane G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome; their amino acid sequences form the largest database for protein-membrane interactions. While there are now structural data on the heptad transmembrane structures of representatives of several heptad families, here we show how fresh insights into global and some local chemical trends in GPCR properties can be obtained accurately from sequences alone, especially by algebraically separating the extracellular and cytoplasmic loops from transmembrane segments. The global mediation of long-range water-protein interactions occurs in conjunction with modulation of these interactions by roughened interfaces. Hydropathic roughening profiles are defined here solely in terms of amino acid sequences, and knowledge of protein coordinates is not required. Roughening profiles both for GPCR and some simpler protein families display accurate and transparent connections to protein functionality, and identify natural length scales for protein functionality.

  10. GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

    PubMed

    Wu, Chia-Shan; Chen, Hongmei; Sun, Hao; Zhu, Jie; Jew, Chris P; Wager-Miller, James; Straiker, Alex; Spencer, Corinne; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2013-01-01

    The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

  11. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  12. Ensemble Activation of G-Protein -Coupled Receptors Revealed by Small-Angle Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Chu, Xiang-Qiang; Perera, Suchithranga; Shrestha, Utsab; Chawla, Udeep; Struts, Andrey; Qian, Shuo; Brown, Michael

    2014-03-01

    Rhodopsin is a G-protein -coupled receptor (GPCR) involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving equilibrium between inactive Meta-I and active Meta-II states. Does photoactivation lead to a single Meta-II conformation, or do substates exist as described by an ensemble-activation mechanism (EAM)? We use small-angle neutron scattering (SANS) to investigate conformational changes in rhodopsin-detergent and rhodopsin-lipid complexes upon photoactivation. Meta-I state is stabilized in CHAPS-solubilized rhodopsin, while Meta-II is trapped in DDM-solubilized rhodopsin. SANS data are acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin has more conformational flexibility in DDM micelles compared to CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, time-resolved SANS enables study of the time-dependent structural transitions between Meta-I and Meta-II, which is crucial to understanding the ensemble-based activation.

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

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    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.

  14. Drug-target residence time--a case for G protein-coupled receptors.

    PubMed

    Guo, Dong; Hillger, Julia M; IJzerman, Adriaan P; Heitman, Laura H

    2014-07-01

    A vast number of marketed drugs act on G protein-coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug-target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on-target side effects. Hence, the compounds' kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug-target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug-target RT.

  15. On the role of G-protein coupled receptors in cell volume regulation.

    PubMed

    Vázquez-Juárez, Erika; Ramos-Mandujano, Gerardo; Hernández-Benítez, Reyna; Pasantes-Morales, Herminia

    2008-01-01

    Cell volume is determined genetically for each cell lineage, but it is not a static feature of the cell. Intracellular volume is continuously challenged by metabolic reactions, uptake of nutrients, intracellular displacement of molecules and organelles and generation of ionic gradients. Moreover, recent evidence raises the intriguing possibility that changes in cell volume act as signals for basic cell functions such as proliferation, migration, secretion and apoptosis. Cells adapt to volume increase by a complex, dynamic process resulting from the concerted action of volume sensing mechanisms and intricate signaling chains, directed to initiate the multiple adaptations demanded by a change in cell volume, among others adhesion reactions, membrane and cytoskeleton remodeling, and activation of the osmolyte pathways leading to reestablish the water balance between extracellular/intracellular or intracellular/intracellular compartments. In multicellular organisms, a continuous interaction with the external milieu is fundamental for the dynamics of the cell. It is in this sense that the recent surge of interest about the influence on cell volume control by the most extended family of signaling elements, the G proteins, acquires particular importance. As here reviewed, a large variety of G-protein coupled receptors (GPCRs) are involved in this interplay with cell volume regulatory mechanisms, which amplifies and diversifies the volume-elicited signaling chains, providing a variety of routes towards the multiple effectors related to cell volume changes.

  16. Isotopic labeling of mammalian G protein-coupled receptors (GPCRs) heterologously expressed in Caenorhabditis elegans*

    PubMed Central

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-01-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack posttranslational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the ‘test’ GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  17. G-protein coupled receptor assays: to measure affinity or efficacy that is the question.

    PubMed

    Williams, C; Sewing, A

    2005-06-01

    Cell-based assays have always played an important role in the pharmaceutical industry, providing information about the functional effects of compounds. These functional assays have traditionally accompanied facile biochemical high throughput screening programmes, being applied as secondary assays in the later stages of lead development. However, with the disappointing reality that there is not likely to be a plethora of novel, druggable targets in the post-genomic era, the role of cell-based assays in drug discovery is beginning to change. Competition to develop the "best" agents for well established targets and find more effective ways of identifying "novel" agents is driving the industry towards a "quality" versus "quantity" approach. Advances in genetic engineering, automation compatible functional assay technologies and the introduction of more sophisticated robotic systems, have facilitated the application of cell-based assays to primary screening. However, despite some apparent success to move these assays into the routine "toolbox" for high throughput screening, certain preconceptions and concerns about cell-based assays persist and the subject remains a topic of much debate. Here we use examples from the screening portfolio at Pfizer, Sandwich, to discuss the practical and theoretical considerations of employing cell-based assays in HTS with a focus on G-protein coupled receptors.

  18. G protein-coupled receptor internalization assays in the high-content screening format.

    PubMed

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  19. Epigenetic regulation of G protein coupled receptor signaling and its implications in psychiatric disorders.

    PubMed

    Dogra, Shalini; Sona, Chandan; Kumar, Ajeet; Yadav, Prem N

    2016-08-01

    G protein-coupled receptors (GPCRs) act as a relay center through which extracellular signals, in the form of neurotransmitters or therapeutics, are converted into an intracellular response, which ultimately shapes the overall response at the tissue and behavioral level. Remarkably in similar ways, epigenetic mechanisms also modulate the expression pattern of a large number of genes in response to the dynamic environment inside and outside of the body, and consequently overall response. Emerging evidences from the pharmacogenomics and preclinical studies clearly suggest that these two distinct mechanisms criss-cross each other in several neurological disorders. At one hand such cross-talks between two distinct mechanisms make disease etiology more challenging to understand, while on the other hand if dealt appropriately, such situations might provide an opportunity to find novel druggable target and strategy for the treatment of complex diseases. In this review article, we have summarized and highlighted the main findings that tie epigenetic mechanisms to GPCR mediated signaling in the pathophysiology of central nervous system (CNS) disorders, including depression, addiction and pain. PMID:27046448

  20. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    PubMed Central

    Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

  1. Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin.

    PubMed

    Yan, Elsa C Y; Kazmi, Manija A; Ganim, Ziad; Hou, Jian-Min; Pan, Douhai; Chang, Belinda S W; Sakmar, Thomas P; Mathies, Richard A

    2003-08-01

    The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110-Cys187); this process may be a general element of G protein-coupled receptor activation.

  2. Lysophosphatidylinositol: a novel link between ABC transporters and G-protein-coupled receptors.

    PubMed

    Ruban, Emily L; Ferro, Riccardo; Arifin, Syamsul Ahmad; Falasca, Marco

    2014-10-01

    Lysophosphatidylinositol (LPI) is a well-known bioactive lipid that is able to activate signalling cascades relevant to cell proliferation, migration, survival and tumorigenesis. Our previous work suggested that LPI is involved in cancer progression since it can be released in the medium of Ras-transformed fibroblasts and can function as an autocrine modulator of cell growth. Different research groups have established that LPI is the specific and functional ligand for G-protein-coupled receptor 55 (GPR55) and that this GPR55-LPI axis is able to activate signalling cascades that are relevant for different cell functions. Work in our laboratory has recently unravelled an autocrine loop, by which LPI synthesized by cytosolic phospholipase A₂ (cPLA₂) is pumped out of the cell by ATP-binding cassette (ABC) transporter C1 (ABCC1)/multidrug resistance protein 1 (MRP1), initiating a signalling cascade downstream of GPR55. Our current work suggests that blockade of this pathway may represent a novel strategy to inhibit cancer cell proliferation. PMID:25233417

  3. GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol, Plays a Role in Motor Coordination

    PubMed Central

    Wu, Chia-Shan; Chen, Hongmei; Sun, Hao; Zhu, Jie; Jew, Chris P.; Wager-Miller, James; Straiker, Alex; Spencer, Corinne; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2013-01-01

    The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior. PMID:23565223

  4. G protein-coupled receptors as therapeutic targets for multiple sclerosis

    PubMed Central

    Du, Changsheng; Xie, Xin

    2012-01-01

    G protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants. They are considered as the most successful therapeutic targets for a broad spectrum of diseases. Multiple sclerosis (MS) is an inflammatory disease that is characterized by immune-mediated demyelination and degeneration of the central nervous system (CNS). It is the leading cause of non-traumatic disability in young adults. Great progress has been made over the past few decades in understanding the pathogenesis of MS. Numerous data from animal and clinical studies indicate that many GPCRs are critically involved in various aspects of MS pathogenesis, including antigen presentation, cytokine production, T-cell differentiation, T-cell proliferation, T-cell invasion, etc. In this review, we summarize the recent findings regarding the expression or functional changes of GPCRs in MS patients or animal models, and the influences of GPCRs on disease severity upon genetic or pharmacological manipulations. Hopefully some of these findings will lead to the development of novel therapies for MS in the near future. PMID:22664908

  5. Interacting residues in an activated state of a G protein-coupled receptor.

    PubMed

    Lee, Yong-Hun; Naider, Fred; Becker, Jeffrey M

    2006-01-27

    Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs. PMID:16314417

  6. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119

    PubMed Central

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2016-01-01

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor. PMID:24184668

  7. G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Barton, Matthias; Prossnitz, Eric R

    2015-10-01

    Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common causes of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase (COX)-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A(2) were determined in human endothelial cells stimulated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). Moreover, Gper-deficient (Gper(-/-)) and WT mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, contractions to acetylcholine-stimulated endothelial vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by twofold. Ovariectomy also augmented prostanoid-dependent contractions by twofold in WT mice but had no additional effect in Gper(-/-) mice. These contractions were blocked by the COX inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor l-N(G)-nitroarginine methyl ester. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to

  8. Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells.

    PubMed

    Scott, Daniel J; Plückthun, Andreas

    2013-02-01

    G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million individual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies.

  9. The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

    PubMed

    Moreno-Ulloa, Aldo; Mendez-Luna, David; Beltran-Partida, Ernesto; Castillo, Carmen; Guevara, Gustavo; Ramirez-Sanchez, Israel; Correa-Basurto, José; Ceballos, Guillermo; Villarreal, Francisco

    2015-10-01

    We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection.

  10. G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Barton, Matthias; Prossnitz, Eric R.

    2016-01-01

    Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common cause of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A2 were determined in human endothelial cells stimulated by the pro-inflammatory cytokine TNF-α. Moreover, Gper-deficient (Gper−/−) and wild-type mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, endothelium-dependent contractions to acetylcholine-stimulated vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by 2-fold. Ovariectomy also augmented prostanoid-dependent contractions by 2-fold in wild-type mice, but had no additional effect in Gper−/− mice. These contractions were blocked by the cyclooxygenase inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor L-NAME. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to treat increased prostanoid

  11. The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

    PubMed

    Moreno-Ulloa, Aldo; Mendez-Luna, David; Beltran-Partida, Ernesto; Castillo, Carmen; Guevara, Gustavo; Ramirez-Sanchez, Israel; Correa-Basurto, José; Ceballos, Guillermo; Villarreal, Francisco

    2015-10-01

    We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection. PMID:26303816

  12. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  13. The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.

    PubMed

    Küffer, Alexander; Lakkaraju, Asvin K K; Mogha, Amit; Petersen, Sarah C; Airich, Kristina; Doucerain, Cédric; Marpakwar, Rajlakshmi; Bakirci, Pamela; Senatore, Assunta; Monnard, Arnaud; Schiavi, Carmen; Nuvolone, Mario; Grosshans, Bianka; Hornemann, Simone; Bassilana, Frederic; Monk, Kelly R; Aguzzi, Adriano

    2016-08-25

    Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options. PMID:27501152

  14. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  15. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors.

    PubMed

    Homan, Kristoff T; Larimore, Kelly M; Elkins, Jonathan M; Szklarz, Marta; Knapp, Stefan; Tesmer, John J G

    2015-01-16

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  16. G Protein-Coupled Receptor 43 Modulates Neutrophil Recruitment during Acute Inflammation

    PubMed Central

    Nicholls, Alyce J.; Oliveira, Ana Carolina; Mason, Linda J.; Binge, Lauren; Mackay, Charles R.; Wong, Connie H. Y.

    2016-01-01

    Fermentation of dietary fibre in the gut yields large amounts of short chain fatty acids (SCFAs). SCFAs can impart biological responses in cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, which suggests that the actions of GPR43 and dietary fibre intake may affect neutrophil recruitment during inflammatory responses in vivo. Using intravital imaging of the small intestine, we found greater intravascular neutrophil rolling and adhesion in Gpr43−/−mice in response to LPS at 1 h. After 4 h of LPS challenge, the intravascular rolling velocity of GPR43-deficient neutrophils was reduced significantly and increased numbers of neutrophils were found in the lamina propria of Gpr43−/−mice. Additionally, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following fMLP challenge. The fMLP-induced neutrophil migration was significantly suppressed in wildtype mice that were treated with acetate, but not in Gpr43−/−mice, strongly suggesting a role for SCFAs in modulating neutrophil migration via GPR43. Indeed, neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour compared to normal chow-fed wildtype mice. Interestingly, this elevated migration could also be reproduced through simple transfer of a no fibre microbiota into germ-free mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Therefore, GPR43 and a microbiota composition that allows for SCFA production function to modulate neutrophil recruitment during inflammatory responses. PMID:27658303

  17. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

    PubMed

    Hamann, Jörg; Aust, Gabriela; Araç, Demet; Engel, Felix B; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A; Harty, Breanne L; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C; Monk, Kelly R; Peeters, Miriam C; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schiöth, Helgi B

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

  18. G protein-coupled receptors: signalling and regulation by lipid agonists for improved glucose homoeostasis.

    PubMed

    Moran, Brian M; Flatt, Peter R; McKillop, Aine M

    2016-04-01

    G protein-coupled receptors (GPCRs) play a pivotal role in cell signalling, controlling many processes such as immunity, growth, cellular differentiation, neurological pathways and hormone secretions. Fatty acid agonists are increasingly recognised as having a key role in the regulation of glucose homoeostasis via stimulation of islet and gastrointestinal GPCRs. Downstream cell signalling results in modulation of the biosynthesis, secretion, proliferation and anti-apoptotic pathways of islet and enteroendocrine cells. GPR40 and GPR120 are activated by long-chain fatty acids (>C12) with both receptors coupling to the Gαq subunit that activates the Ca(2+)-dependent pathway. GPR41 and GPR43 are stimulated by short-chain fatty acids (C2-C5), and activation results in binding to Gαi that inhibits the adenylyl cyclase pathway attenuating cAMP production. In addition, GPR43 also couples to the Gαq subunit augmenting intracellular Ca(2+) and activating phospholipase C. GPR55 is specific for cannabinoid endogenous agonists (endocannabinoids) and non-cannabinoid fatty acids, which couples to Gα12/13 and Gαq proteins, leading to enhancing intracellular Ca(2+), extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and Rho kinase. GPR119 is activated by fatty acid ethanolamides and binds to Gαs utilising the adenylate cyclase pathway, which is dependent upon protein kinase A. Current research indicates that GPCR therapies may be approved for clinical use in the near future. This review focuses on the recent advances in preclinical diabetes research in the signalling and regulation of GPCRs on islet and enteroendocrine cells involved in glucose homoeostasis.

  19. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    EPA Science Inventory

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

    Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

    Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  20. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis. PMID:22100731

  1. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.

  2. Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54.

    PubMed

    Messager, Sophie; Chatzidaki, Emmanouella E; Ma, Dan; Hendrick, Alan G; Zahn, Dirk; Dixon, John; Thresher, Rosemary R; Malinge, Isabelle; Lomet, Didier; Carlton, Mark B L; Colledge, William H; Caraty, Alain; Aparicio, Samuel A J R

    2005-02-01

    We have recently described a molecular gatekeeper of the hypothalamic-pituitary-gonadal axis with the observation that G protein-coupled receptor 54 (GPR54) is required in mice and men for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion to occur. In the present study, we investigate the possible central mode of action of GPR54 and kisspeptin ligand. First, we show that GPR54 transcripts are colocalized with gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, may act directly on these neurons. Next, we show that GnRH neurons seem anatomically normal in gpr54-/- mice, and that they show projections to the median eminence, which demonstrates that the hypogonadism in gpr54-/- mice is not due to an abnormal migration of GnRH neurons (as occurs with KAL1 mutations), but that it is more likely due to a lack of GnRH release or absence of GnRH neuron stimulation. We also show that levels of kisspeptin injected i.p., which stimulate robust LH and FSH release in wild-type mice, have no effect in gpr54-/- mice, and therefore that kisspeptin acts directly and uniquely by means of GPR54 signaling for this function. Finally, we demonstrate by direct measurement, that the central administration of kisspeptin intracerebroventricularly in sheep produces a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin on the hypothalamo-pituitary-gonadal axis occurs directly at the level of GnRH release. The localization and GnRH release effects of kisspeptin thus define GPR54 as a major control point in the reproductive axis and suggest kisspeptin to be a neurohormonal effector.

  3. Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor.

    PubMed

    Gao, Zhan-Guo; Balasubramanian, Ramachandran; Kiselev, Evgeny; Wei, Qiang; Jacobson, Kenneth A

    2014-08-01

    G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N⁶-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic β cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.

  4. G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker.

    PubMed

    Zheng, Xiaoyi; Chang, Frank; Zhang, Xinmin; Rothman, Vicki L; Tuszynski, George P

    2012-08-01

    Using an innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and late stage breast tumors (Tuszynski, G.P. et al., 2011). In this study we further investigated the significance of GASP-1 as a tumor marker by investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast cancers as compared to their respective normal tissues as assessed by immunohistochemical staining of tissue arrays using a "peptide specific" GASP-1 antibody. We found that across these cancers, GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue. The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the brain tumors. Using a sensitive "competitive ELISA" for GASP-1, we found that sera from patients with brain, liver, breast and lung cancers expressed 4-7 fold more GASP-1 peptide than sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for development of cancer therapeutics. PMID:22483848

  5. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    PubMed

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system.

  6. Chemogenomics knowledgebased polypharmacology analyses of drug abuse related G-protein coupled receptors and their ligands.

    PubMed

    Xie, Xiang-Qun; Wang, Lirong; Liu, Haibin; Ouyang, Qin; Fang, Cheng; Su, Weiwei

    2014-01-01

    Drug abuse (DA) and addiction is a complex illness, broadly viewed as a neurobiological impairment with genetic and environmental factors that influence its development and manifestation. Abused substances can disrupt the activity of neurons by interacting with many proteins, particularly G-protein coupled receptors (GPCRs). A few medicines that target the central nervous system (CNS) can also modulate DA related proteins, such as GPCRs, which can act in conjunction with the controlled psychoactive substance(s) and increase side effects. To fully explore the molecular interaction networks that underlie DA and to effectively modulate the GPCRs in these networks with small molecules for DA treatment, we built a drug-abuse domain specific chemogenomics knowledgebase (DA-KB) to centralize the reported chemogenomics research information related to DA and CNS disorders in an effort to benefit researchers across a broad range of disciplines. We then focus on the analysis of GPCRs as many of them are closely related with DA. Their distribution in human tissues was also analyzed for the study of side effects caused by abused drugs. We further implement our computational algorithms/tools to explore DA targets, DA mechanisms and pathways involved in polydrug addiction and to explore polypharmacological effects of the GPCR ligands. Finally, the polypharmacology effects of GPCRs-targeted medicines for DA treatment were investigated and such effects can be exploited for the development of drugs with polypharmacophore for DA intervention. The chemogenomics database and the analysis tools will help us better understand the mechanism of drugs abuse and facilitate to design new medications for system pharmacotherapy of DA. PMID:24567719

  7. Mechanisms of G protein-coupled estrogen receptor-mediated spinal nociception

    PubMed Central

    Deliu, Elena; Brailoiu, G. Cristina; Arterburn, Jeffrey B.; Oprea, Tudor I.; Benamar, Khalid; Dun, Nae J.; Brailoiu, Eugen

    2012-01-01

    Human and animal studies suggest estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pro-nociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER) activation. Membrane depolarization, increases in cytosolic calcium and reactive oxygen species (ROS) levels are markers of neuronal activation, underlying pain sensitization in the spinal cord. Using behavioral, electrophysiological and fluorescent imaging studies, we evaluated GPER involvement in spinal nociceptive processing. Intrathecal challenging of mice with the GPER agonist G-1 results in pain-related behaviors. GPER antagonism with G15 reduces the G-1 induced response. Electrophysiological recordings from superficial dorsal horn neurons indicate neuronal membrane depolarization upon G-1 application, which is G15 sensitive. In cultured spinal sensory neurons G-1 increases intracellular calcium concentration and induces mitochondrial and cytosolic ROS accumulation. In the presence of G15, G-1 does not elicit the calcium and ROS responses, confirming specific GPER involvement in this process. Following G-1 intracellular microinjections, cytosolic calcium concentration elevates faster and with higher amplitude compared to extracellular exposure, suggesting subcellular GPER functionality. Thus, GPER activation results in spinal nociception, and the downstream mechanisms involve cytosolic calcium increase, ROS accumulation and neuronal membrane depolarization. Perspective Our results suggest that GPER modulates pain processing in spinal sensory neurons via cytosolic calcium increase and ROS accumulation. These findings extend the current knowledge on GPER involvement in physiology and disease, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the underlying mechanisms. PMID:22858342

  8. [Role of orphan G protein-coupled receptor 55 in diabetic gastroparesis in mice].

    PubMed

    Lin, Xu-Hong; Wei, Dan-Dan; Wang, Hui-Chao; Wang, Bin; Bai, Chun-Yang; Wang, Ya-Qiang; Li, Guo-En; Li, Hui-Ping; Ren, Xue-Qun

    2014-06-25

    The aim of the present study was to explore the role of orphan G protein-coupled receptor 55 (GPR55) in diabetic gastroparesis (DG). Streptozotocin (STZ) was used to mimic the DG model, and the body weight and blood glucose concentration were tested 4 weeks after STZ injection (i.p.). Electrogastrogram and phenolsulfonphthalein test were used for detecting gastric emptying. Motilin (MTL), gastrin (GAS), vasoactive intestinal peptide (VIP), and somatostatin (SS) levels in plasma were determined using radioimmunology. Real-time PCR and Western blot were applied to identify the expression of GPR55 in gastric tissue, and immunohistochemistry was used to detect the distribution. The effect of lysophosphatidylinositol (LPI), an agonist of GPR55, was observed. STZ mice showed increased blood glucose concentration, lower body weight, decreased amplitude of slow wave, and delayed gastric emptying. LPI antagonized these effects of STZ. Compared to the control group, STZ caused significant decreases of MTL and GAS levels (P < 0.01), as well as increases of SS and VIP levels (P < 0.01). The changes of these hormones induced by STZ were counteracted when using LPI. GPR55 located in mice stomach, and it was up-regulated in DG. Although LPI showed no effects on the distribution and expression of GPR55 in normal mice, it could inhibit STZ-induced GPR55 up-regulation. These results suggest GPR55 is involved in the regulation of gastric movement of DG, and may serve as a new target of DG treatment. LPI, an agonist of GPR55, can protect against STZ-induced DG, and the mechanism may involve the change of GPR55 expression and modification of gastrointestinal movement regulating hormones. PMID:24964851

  9. Chemogenomics knowledgebased polypharmacology analyses of drug abuse related G-protein coupled receptors and their ligands

    PubMed Central

    Xie, Xiang-Qun; Wang, Lirong; Liu, Haibin; Ouyang, Qin; Fang, Cheng; Su, Weiwei

    2013-01-01

    Drug abuse (DA) and addiction is a complex illness, broadly viewed as a neurobiological impairment with genetic and environmental factors that influence its development and manifestation. Abused substances can disrupt the activity of neurons by interacting with many proteins, particularly G-protein coupled receptors (GPCRs). A few medicines that target the central nervous system (CNS) can also modulate DA related proteins, such as GPCRs, which can act in conjunction with the controlled psychoactive substance(s) and increase side effects. To fully explore the molecular interaction networks that underlie DA and to effectively modulate the GPCRs in these networks with small molecules for DA treatment, we built a drug-abuse domain specific chemogenomics knowledgebase (DA-KB) to centralize the reported chemogenomics research information related to DA and CNS disorders in an effort to benefit researchers across a broad range of disciplines. We then focus on the analysis of GPCRs as many of them are closely related with DA. Their distribution in human tissues was also analyzed for the study of side effects caused by abused drugs. We further implement our computational algorithms/tools to explore DA targets, DA mechanisms and pathways involved in polydrug addiction and to explore polypharmacological effects of the GPCR ligands. Finally, the polypharmacology effects of GPCRs-targeted medicines for DA treatment were investigated and such effects can be exploited for the development of drugs with polypharmacophore for DA intervention. The chemogenomics database and the analysis tools will help us better understand the mechanism of drugs abuse and facilitate to design new medications for system pharmacotherapy of DA. PMID:24567719

  10. G protein-coupled estrogen receptor 1-mediated effects in the rat myometrium.

    PubMed

    Tica, Andrei A; Dun, Erica C; Tica, Oana S; Gao, Xin; Arterburn, Jeffrey B; Brailoiu, G Cristina; Oprea, Tudor I; Brailoiu, Eugen

    2011-11-01

    G protein-coupled estrogen receptor 1 (GPER), also named GPR30, has been previously identified in the female reproductive system. In this study, GPER expression was found in the female rat myometrium by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Using GPER-selective ligands, we assessed the effects of the GPER activation on resting membrane potential and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in rat myometrial cells, as well as on contractility of rat uterine strips. G-1, a specific GPER agonist, induced a concentration-dependent depolarization and increase in [Ca(2+)](i) in myometrial cells. The depolarization was abolished in Na(+)-free saline. G-1-induced [Ca(2+)](i) increase was markedly decreased by nifedipine, a L-type Ca(2+) channel blocker, by Ca(2+)-free or Na(+)-free saline. Intracellular administration of G-1 produced a faster and transitory increase in [Ca(2+)](i), with a higher amplitude than that induced by extracellular application, supporting an intracellular localization of the functional GPER in myometrial cells. Depletion of internal Ca(2+) stores with thapsigargin produced a robust store-activated Ca(2+) entry; the Ca(2+) response to G-1 was similar to the constitutive Ca(2+) entry and did not seem to involve store-operated Ca(2+) entry. In rat uterine strips, administration of G-1 increased the frequency and amplitude of contractions and the area under the contractility curve. The effects of G-1 on membrane potential, [Ca(2+)](i), and uterine contractility were prevented by pretreatment with G-15, a GPER antagonist, further supporting the involvement of GPER in these responses. Taken together, our results indicate that GPER is expressed and functional in rat myometrium. GPER activation produces depolarization, elevates [Ca(2+)](i) and increases contractility in myometrial cells.

  11. Discovery and mapping of ten novel G protein-coupled receptor genes.

    PubMed

    Lee, D K; Nguyen, T; Lynch, K R; Cheng, R; Vanti, W B; Arkhitko, O; Lewis, T; Evans, J F; George, S R; O'Dowd, B F

    2001-09-01

    We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed

  12. Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.

    PubMed

    Ray-Saha, Sarmistha; Huber, Thomas; Sakmar, Thomas P

    2014-03-01

    We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes. PMID:24490954

  13. G protein-coupled receptor signalling in astrocytes in health and disease: a focus on metabotropic glutamate receptors.

    PubMed

    Bradley, Sophie J; Challiss, R A John

    2012-08-01

    Work published over the past 10-15 years has caused the neuroscience community to engage in a process of constant re-evaluation of the roles of glial cells in the mammalian central nervous system. Recent emerging evidence suggests that, in addition to carrying out various homeostatic functions within the CNS, astrocytes can also engage in a two-way dialogue with neurons. Astrocytes possess many of the receptors, and some of the ion channels, present in neurons endowing them with an ability to sense and respond to an array of neuronal signals. In addition, an expanding number of small molecules and proteins have been shown to be released by astrocytes in both health and disease. In this commentary we will highlight advances in our understanding of G protein-coupled receptor signalling in astrocytes, with a particular emphasis on metabotropic glutamate (mGlu) receptors. Discussion will focus on the major mGlu receptors expressed in astrocytes, mGlu3 and mGlu5, how these receptors can influence different aspects of astrocyte physiology, and how signalling by these G protein-coupled receptors might change under pathophysiological circumstances. PMID:22531220

  14. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    PubMed

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  15. G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

    PubMed

    Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

    2011-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.

  16. [Antinociceptive effect of docosahexaenoic acid (DHA) through long fatty acid receptor G protein-coupled receptor 40 (GPR40)].

    PubMed

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Tokuyama, Shogo

    2014-01-01

    Fatty acids, one class of essential nutrients for humans, are an important source of energy and an essential component of cell membranes. They also function as signal transduction molecules in a variety of biological phenomena. The important functional role of fatty acids in both onset and suppression of pain has become increasingly apparent in recent years. Recently, we have also demonstrated that the release of an endogenous opioid peptide, β-endorphin, plays an important role in the induction of docosahexaenoic acid (DHA)-induced antinociception. It is well known that fatty acids affect intracellular and intercellular signaling as well as the membrane fluidity of neurons. In addition to intracellular actions, unbound free fatty acids (FFAs) can also carry out extracellular signaling by stimulating the G protein-coupled receptor (GPCR). Among these receptors, G protein-coupled receptor 40 (GPR40) has been reported to be activated by long-chain fatty acids such as DHA, eicosapentaenoic acid (EPA) and arachidonic acid. In the peripheral area, GPR40 is preferentially expressed in pancreatic β-cells and is known to relate to the secretion of hormone and peptides. On the other hand, even though this receptor is widely distributed in the central nervous system, reports studying the role and functions of GPR40 in the brain have not been found. In this review, we summarize the findings of our recent study about the long-chain fatty acid receptor GPR40 as a novel pain regulatory system. PMID:24584021

  17. G-protein Coupled Receptor 30 Interacts with Receptor Activity Modifying Protein 3 and Confers Sex-Dependent Cardioprotection

    PubMed Central

    Lenhart, Patricia M.; Broselid, Stefan; Barrick, Cordelia J.; Leeb-Lundberg, L.M. Fredrik; Caron, Kathleen M.

    2013-01-01

    Receptor activity modifying protein 3 (RAMP3) is a single pass transmembrane protein known to interact with and affect the trafficking of several G-protein coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with G-protein coupled receptor 30 (GPR30), also known as G-protein estrogen receptor 1 (GPER1). GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Utilizing bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3- and sex-dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo, and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  18. Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach

    PubMed Central

    Ivetac, Anthony; Andrew McCammon, J

    2010-01-01

    To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the β1 (β1AR) and β2 (β2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify ‘hot spots’ with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 μs. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies. PMID:20626410

  19. FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans.

    PubMed

    Nelson, Matthew D; Janssen, Tom; York, Neil; Lee, Kun He; Schoofs, Liliane; Raizen, David M

    2015-01-01

    Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence. PMID:26571132

  20. FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans

    PubMed Central

    York, Neil; Lee, Kun He; Schoofs, Liliane; Raizen, David M.

    2015-01-01

    Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence. PMID:26571132

  1. G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection.

    PubMed

    Lenhart, Patricia M; Broselid, Stefan; Barrick, Cordelia J; Leeb-Lundberg, L M Fredrik; Caron, Kathleen M

    2013-01-01

    Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  2. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  3. Analysis of Drug Design for a Selection of G Protein-Coupled Neuro- Receptors Using Neural Network Techniques.

    PubMed

    Agerskov, Claus; Mortensen, Rasmus M; Bohr, Henrik G

    2015-01-01

    A study is presented on how well possible drug-molecules can be predicted with respect to their function and binding to a selection of neuro-receptors by the use of artificial neural networks. The ligands investigated in this study are chosen to be corresponding to the G protein-coupled receptors µ-opioid, serotonin 2B (5-HT2B) and metabotropic glutamate D5. They are selected due to the availability of pharmacological drug-molecule binding data for these receptors. Feedback and deep belief artificial neural network architectures (NNs) were chosen to perform the task of aiding drugdesign. This is done by training on structural features, selected using a "minimum redundancy, maximum relevance"-test, and testing for successful prediction of categorized binding strength. An extensive comparison of the neural network performances was made in order to select the optimal architecture. Deep belief networks, trained with greedy learning algorithms, showed superior performance in prediction over the simple feedback NNs. The best networks obtained scores of more than 90 % accuracy in predicting the degree of binding drug molecules to the mentioned receptors and with a maximal Matthew`s coefficient of 0.925. The performance of 8 category networks (8 output classes for binding strength) obtained a prediction accuracy of above 60 %. After training the networks, tests were done on how well the systems could be used as an aid in designing candidate drug molecules. Specifically, it was shown how a selection of chemical characteristics could give the lowest observed IC50 values, meaning largest bio-effect pr. nM substance, around 0.03-0.06 nM. These ligand characteristics could be total number of atoms, their types etc. In conclusion, deep belief networks trained on drug-molecule structures were demonstrated as powerful computational tools, able to aid in drug-design in a fast and cheap fashion, compared to conventional pharmacological techniques.

  4. Analysis of Drug Design for a Selection of G Protein-Coupled Neuro- Receptors Using Neural Network Techniques.

    PubMed

    Agerskov, Claus; Mortensen, Rasmus M; Bohr, Henrik G

    2015-01-01

    A study is presented on how well possible drug-molecules can be predicted with respect to their function and binding to a selection of neuro-receptors by the use of artificial neural networks. The ligands investigated in this study are chosen to be corresponding to the G protein-coupled receptors µ-opioid, serotonin 2B (5-HT2B) and metabotropic glutamate D5. They are selected due to the availability of pharmacological drug-molecule binding data for these receptors. Feedback and deep belief artificial neural network architectures (NNs) were chosen to perform the task of aiding drugdesign. This is done by training on structural features, selected using a "minimum redundancy, maximum relevance"-test, and testing for successful prediction of categorized binding strength. An extensive comparison of the neural network performances was made in order to select the optimal architecture. Deep belief networks, trained with greedy learning algorithms, showed superior performance in prediction over the simple feedback NNs. The best networks obtained scores of more than 90 % accuracy in predicting the degree of binding drug molecules to the mentioned receptors and with a maximal Matthew`s coefficient of 0.925. The performance of 8 category networks (8 output classes for binding strength) obtained a prediction accuracy of above 60 %. After training the networks, tests were done on how well the systems could be used as an aid in designing candidate drug molecules. Specifically, it was shown how a selection of chemical characteristics could give the lowest observed IC50 values, meaning largest bio-effect pr. nM substance, around 0.03-0.06 nM. These ligand characteristics could be total number of atoms, their types etc. In conclusion, deep belief networks trained on drug-molecule structures were demonstrated as powerful computational tools, able to aid in drug-design in a fast and cheap fashion, compared to conventional pharmacological techniques. PMID:26463104

  5. Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily*

    PubMed Central

    Roth, Bryan L.; Kroeze, Wesley K.

    2015-01-01

    G-protein-coupled receptors (GPCRs) are frequent and fruitful targets for drug discovery and development, as well as being off-targets for the side effects of a variety of medications. Much of the druggable non-olfactory human GPCR-ome remains under-interrogated, and we present here various approaches that we and others have used to shine light into these previously dark corners of the human genome. PMID:26100629

  6. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  7. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

    PubMed

    Chu, Jiaojiao; Zhang, Hangxiang; Huang, Xiuqing; Lin, Yajun; Shen, Tao; Chen, Beidong; Man, Yong; Wang, Shu; Li, Jian

    2013-01-01

    Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  8. Advances in the Development and Application of Computational Methodologies for Structural Modeling of G-Protein Coupled Receptors

    PubMed Central

    Mobarec, Juan Carlos

    2009-01-01

    Background Despite the large amount of experimental data accumulated in the past decade on G-protein coupled receptor (GPCR) structure and function, understanding of the molecular mechanisms underlying GPCR signaling is still far from being complete, thus impairing the design of effective and selective pharmaceuticals. Objective Understanding of GPCR function has been challenged even further by more recent experimental evidence that several of these receptors are organized in the cell membrane as homo- or hetero-oligomers, and that they may exhibit unique pharmacological properties. Given the complexity of these new signaling systems, researcher’s efforts are turning increasingly to molecular modeling, bioinformatics and computational simulations for mechanistic insights of GPCR functional plasticity. Methods We review here current advances in the development and application of computational approaches to improve prediction of GPCR structure and dynamics, thus enhancing current understanding of GPCR signaling. Results/Conclusions Models resulting from use of these computational approaches further supported by experiments are expected to help elucidate the complex allosterism that propagates through GPCR complexes, ultimately aiming at successful structure-based rational drug design. PMID:19672320

  9. Unraveling the molecular architecture of a G protein-coupled receptor/β-arrestin/Erk module complex

    PubMed Central

    Bourquard, Thomas; Landomiel, Flavie; Reiter, Eric; Crépieux, Pascale; Ritchie, David W.; Azé, Jérôme; Poupon, Anne

    2015-01-01

    β-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of β-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/β-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the β-arrestin/Raf1 and the β-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partners, hereby demonstrating the validity of the predicted interaction regions. PMID:26030356

  10. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    PubMed Central

    Wang, Zhixiang

    2016-01-01

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges. PMID:26771606

  11. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

    PubMed

    Chang, Gin-Wen; Hsiao, Cheng-Chih; Peng, Yen-Ming; Vieira Braga, Felipe A; Kragten, Natasja A M; Remmerswaal, Ester B M; van de Garde, Martijn D B; Straussberg, Rachel; König, Gabriele M; Kostenis, Evi; Knäuper, Vera; Meyaard, Linde; van Lier, René A W; van Gisbergen, Klaas P J M; Lin, Hsi-Hsien; Hamann, Jörg

    2016-05-24

    Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells. PMID:27184850

  12. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    SciTech Connect

    Heiber, M.; Marchese, A.; O`Dowd, B.F.

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  13. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    SciTech Connect

    Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro; Matsuda, Kouhei; Asaoka, Yoichi; Nishina, Hiroshi; Nakakura, Takashi; Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu; Tomura, Hideaki

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  14. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

    PubMed Central

    Hermans, E; Challiss, R A

    2001-01-01

    In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders. PMID:11672421

  15. Knockin mice expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor dynamics in vivo.

    PubMed

    Scherrer, Grégory; Tryoen-Tóth, Petra; Filliol, Dominique; Matifas, Audrey; Laustriat, Delphine; Cao, Yu Q; Basbaum, Allan I; Dierich, Andrée; Vonesh, Jean-Luc; Gavériaux-Ruff, Claire; Kieffer, Brigitte L

    2006-06-20

    The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the delta-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knock-in mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design. PMID:16766653

  16. Discovery of selective probes and antagonists for G-protein-coupled receptors FPR/FPRL1 and GPR30.

    PubMed

    Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R; Edwards, Bruce S; Sklar, Larry A

    2009-01-01

    Recent technological advances in flow cytometry provide a versatile platform for high throughput screening of compound libraries coupled with high-content biological testing and drug discovery. The G protein-coupled receptors (GPCRs) constitute the largest class of signaling molecules in the human genome with frequent roles in disease pathogenesis, yet many examples of orphan receptors with unknown ligands remain. The complex biology and potential for drug discovery within this class provide strong incentives for chemical biology approaches seeking to develop small molecule probes to facilitate elucidation of mechanistic pathways and enable specific manipulation of the activity of individual receptors. We have initiated small molecule probe development projects targeting two distinct families of GPCRs: the formylpeptide receptors (FPR/FPRL1) and G protein-coupled estrogen receptor (GPR30). In each case the assay for compound screening involved the development of an appropriate small molecule fluorescent probe, and the flow cytometry platform provided inherently biological rich assays that enhanced the process of identification and optimization of novel antagonists. The contributions of cheminformatics analysis tools, virtual screening, and synthetic chemistry in synergy with the biomolecular screening program have yielded valuable new chemical probes with high binding affinity, selectivity for the targeted receptor, and potent antagonist activity. This review describes the discovery of novel small molecule antagonists of FPR and FPRL1, and GPR30, and the associated characterization process involving secondary assays, cell based and in vivo studies to define the selectivity and activity of the resulting chemical probes.

  17. Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

    PubMed Central

    Berbari, Nicolas F.; Johnson, Andrew D.; Lewis, Jacqueline S.; Askwith, Candice C.

    2008-01-01

    Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein–coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight. PMID:18256283

  18. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells. PMID:25881750

  19. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells.

  20. Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.

    PubMed

    Borroto-Escuela, Dasiel O; Flajolet, Marc; Agnati, Luigi F; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described.

  1. BIOLUMINISCENCE RESONANCE ENERGY TRANSFER (BRET) METHODS TO STUDY G PROTEIN-COUPLED RECEPTOR - RECEPTOR TYROSINE KINASE HETERORECEPTOR COMPLEXES

    PubMed Central

    Borroto-Escuela, Dasiel O.; Flajolet, Marc; Agnati, Luigi F.; Greengard, Paul; Fuxe, Kjell

    2014-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and Receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signalling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signalling molecules. This integrative phenomenon is reciprocal, and can place also RTK signalling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization and down regulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this work we provide an overview of different BRET2 assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described. PMID:24143976

  2. A Hybrid Approach to Structure and Function Modeling of G Protein-Coupled Receptors.

    PubMed

    Latek, Dorota; Bajda, Marek; Filipek, Sławomir

    2016-04-25

    The recent GPCR Dock 2013 assessment of serotonin receptor 5-HT1B and 5-HT2B, and smoothened receptor SMO targets, exposed the strengths and weaknesses of the currently used computational approaches. The test cases of 5-HT1B and 5-HT2B demonstrated that both the receptor structure and the ligand binding mode can be predicted with the atomic-detail accuracy, as long as the target-template sequence similarity is relatively high. On the other hand, the observation of a low target-template sequence similarity, e.g., between SMO from the frizzled GPCR family and members of the rhodopsin family, hampers the GPCR structure prediction and ligand docking. Indeed, in GPCR Dock 2013, accurate prediction of the SMO target was still beyond the capabilities of most research groups. Another bottleneck in the current GPCR research, as demonstrated by the 5-HT2B target, is the reliable prediction of global conformational changes induced by activation of GPCRs. In this work, we report details of our protocol used during GPCR Dock 2013. Our structure prediction and ligand docking protocol was especially successful in the case of 5-HT1B and 5-HT2B-ergotamine complexes for which we provide one of the most accurate predictions. In addition to a description of the GPCR Dock 2013 results, we propose a novel hybrid computational methodology to improve GPCR structure and function prediction. This computational methodology employs two separate rankings for filtering GPCR models. The first ranking is ligand-based while the second is based on the scoring scheme of the recently published BCL method. In this work, we prove that the use of knowledge-based potentials implemented in BCL is an efficient way to cope with major bottlenecks in the GPCR structure prediction. Thereby, we also demonstrate that the knowledge-based potentials for membrane proteins were significantly improved, because of the recent surge in available experimental structures.

  3. Classification of G-protein coupled receptors based on support vector machine with maximum relevance minimum redundancy and genetic algorithm

    PubMed Central

    2010-01-01

    Background Because a priori knowledge about function of G protein-coupled receptors (GPCRs) can provide useful information to pharmaceutical research, the determination of their function is a quite meaningful topic in protein science. However, with the rapid increase of GPCRs sequences entering into databanks, the gap between the number of known sequence and the number of known function is widening rapidly, and it is both time-consuming and expensive to determine their function based only on experimental techniques. Therefore, it is vitally significant to develop a computational method for quick and accurate classification of GPCRs. Results In this study, a novel three-layer predictor based on support vector machine (SVM) and feature selection is developed for predicting and classifying GPCRs directly from amino acid sequence data. The maximum relevance minimum redundancy (mRMR) is applied to pre-evaluate features with discriminative information while genetic algorithm (GA) is utilized to find the optimized feature subsets. SVM is used for the construction of classification models. The overall accuracy with three-layer predictor at levels of superfamily, family and subfamily are obtained by cross-validation test on two non-redundant dataset. The results are about 0.5% to 16% higher than those of GPCR-CA and GPCRPred. Conclusion The results with high success rates indicate that the proposed predictor is a useful automated tool in predicting GPCRs. GPCR-SVMFS, a corresponding executable program for GPCRs prediction and classification, can be acquired freely on request from the authors. PMID:20550715

  4. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors.

    PubMed

    Masuho, Ikuo; Ostrovskaya, Olga; Kramer, Grant M; Jones, Christopher D; Xie, Keqiang; Martemyanov, Kirill A

    2015-12-01

    Members of the heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) family play key roles in many physiological functions and are extensively exploited pharmacologically to treat diseases. Many of the diverse effects of individual GPCRs on cellular physiology are transduced by heterotrimeric G proteins, which are composed of α, β, and γ subunits. GPCRs interact with and stimulate the binding of guanosine triphosphate (GTP) to the α subunit to initiate signaling. Mammalian genomes encode 16 different G protein α subunits, each one of which has distinct properties. We developed a single-platform, optical strategy to monitor G protein activation in live cells. With this system, we profiled the coupling ability of individual GPCRs for different α subunits, simultaneously quantifying the magnitude of the signal and the rates at which the receptors activated the G proteins. We found that individual receptors engaged multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles. Different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists, distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein-coupling profiles of GPCRs, which suggests that their differential abundance may alter signaling outcomes in a cell-specific manner. These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins, coupling to which produces signals with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically. PMID:26628681

  5. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    PubMed

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner. PMID:27424906

  6. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    PubMed

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

  7. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus.

    PubMed

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  8. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus

    PubMed Central

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  9. International Union of Basic and Clinical Pharmacology. XCVII. G Protein-Coupled Estrogen Receptor and Its Pharmacologic Modulators.

    PubMed

    Prossnitz, Eric R; Arterburn, Jeffrey B

    2015-07-01

    Estrogens are critical mediators of multiple and diverse physiologic effects throughout the body in both sexes, including the reproductive, cardiovascular, endocrine, nervous, and immune systems. As such, alterations in estrogen function play important roles in many diseases and pathophysiological conditions (including cancer), exemplified by the lower prevalence of many diseases in premenopausal women. Estrogens mediate their effects through multiple cellular receptors, including the nuclear receptor family (ERα and ERβ) and the G protein-coupled receptor (GPCR) family (GPR30/G protein-coupled estrogen receptor [GPER]). Although both receptor families can initiate rapid cell signaling and transcriptional regulation, the nuclear receptors are traditionally associated with regulating gene expression, whereas GPCRs are recognized as mediating rapid cellular signaling. Estrogen-activated pathways are not only the target of multiple therapeutic agents (e.g., tamoxifen, fulvestrant, raloxifene, and aromatase inhibitors) but are also affected by a plethora of phyto- and xeno-estrogens (e.g., genistein, coumestrol, bisphenol A, dichlorodiphenyltrichloroethane). Because of the existence of multiple estrogen receptors with overlapping ligand specificities, expression patterns, and signaling pathways, the roles of the individual receptors with respect to the diverse array of endogenous and exogenous ligands have been challenging to ascertain. The identification of GPER-selective ligands however has led to a much greater understanding of the roles of this receptor in normal physiology and disease as well as its interactions with the classic estrogen receptors ERα and ERβ and their signaling pathways. In this review, we describe the history and characterization of GPER over the past 15 years focusing on the pharmacology of steroidal and nonsteroidal compounds that have been employed to unravel the biology of this most recently recognized estrogen receptor.

  10. Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway.

    PubMed

    Yuan, Shuguang; Filipek, Slawomir; Palczewski, Krzysztof; Vogel, Horst

    2014-09-09

    Recent crystal structures of G-protein-coupled receptors (GPCRs) have revealed ordered internal water molecules, raising questions about the functional role of those waters for receptor activation that could not be answered by the static structures. Here, we used molecular dynamics simulations to monitor--at atomic and high temporal resolution--conformational changes of central importance for the activation of three prototypical GPCRs with known crystal structures: the adenosine A2A receptor, the β2-adrenergic receptor and rhodopsin. Our simulations reveal that a hydrophobic layer of amino acid residues next to the characteristic NPxxY motif forms a gate that opens to form a continuous water channel only upon receptor activation. The highly conserved tyrosine residue Y(7.53) undergoes transitions between three distinct conformations representative of inactive, G-protein activated and GPCR metastates. Additional analysis of the available GPCR crystal structures reveals general principles governing the functional roles of internal waters in GPCRs.

  11. The G Protein-coupled Receptor P2Y14 Influences Insulin Release and Smooth Muscle Function in Mice*

    PubMed Central

    Meister, Jaroslawna; Le Duc, Diana; Ricken, Albert; Burkhardt, Ralph; Thiery, Joachim; Pfannkuche, Helga; Polte, Tobias; Grosse, Johannes; Schöneberg, Torsten; Schulz, Angela

    2014-01-01

    UDP sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor P2Y14 (GPR105) was found to bind extracellular UDP and UDP sugars. Little is known about the physiological functions of this G protein-coupled receptor. To study its physiological role, we used a gene-deficient mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung, and uterus. Among other phenotypical differences, knock-out mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets, highlighting P2Y14 as a new modulator of proper insulin secretion. PMID:24993824

  12. Multiple Scaffolding Functions of β-Arrestins in the Degradation of G Protein-coupled Receptor Kinase 2*

    PubMed Central

    Nogués, Laura; Salcedo, Alicia; Mayor, Federico; Penela, Petronila

    2011-01-01

    G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts. PMID:21081496

  13. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  14. Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors.

    PubMed

    Hamby, Mary E; Coppola, Giovanni; Ao, Yan; Geschwind, Daniel H; Khakh, Baljit S; Sofroniew, Michael V

    2012-10-17

    Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2ry1, Gnao1, Gng7), but some up (for example, P2ry14, P2ry6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.

  15. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.

    PubMed

    Schütz, Marco; Schöppe, Jendrik; Sedlák, Erik; Hillenbrand, Matthias; Nagy-Davidescu, Gabriela; Ehrenmann, Janosch; Klenk, Christoph; Egloff, Pascal; Kummer, Lutz; Plückthun, Andreas

    2016-02-25

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.

  16. Activation Biosensor for G Protein-Coupled Receptors: A FRET-Based m1 Muscarinic Activation Sensor That Regulates Gq

    PubMed Central

    Chang, Seungwoo; Ross, Elliott M.

    2012-01-01

    We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I Gq-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15–20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to catalyze activation of Gαq, and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by Gq. We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with Gαq, in the context of the known structures of Class I G protein-coupled receptors. The approach should be generally applicable to other Class I receptors which include numerous important drug targets. PMID:23029161

  17. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts

    PubMed Central

    Schütz, Marco; Schöppe, Jendrik; Sedlák, Erik; Hillenbrand, Matthias; Nagy-Davidescu, Gabriela; Ehrenmann, Janosch; Klenk, Christoph; Egloff, Pascal; Kummer, Lutz; Plückthun, Andreas

    2016-01-01

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation. PMID:26911446

  18. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.

    PubMed

    Schütz, Marco; Schöppe, Jendrik; Sedlák, Erik; Hillenbrand, Matthias; Nagy-Davidescu, Gabriela; Ehrenmann, Janosch; Klenk, Christoph; Egloff, Pascal; Kummer, Lutz; Plückthun, Andreas

    2016-01-01

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation. PMID:26911446

  19. The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

    PubMed

    Lee, M J; Evans, M; Hla, T

    1996-05-10

    The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1. PMID:8626678

  20. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. PMID:27378756

  1. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

  2. Adropin acts in brain to inhibit water drinking: potential interaction with the orphan G protein-coupled receptor, GPR19.

    PubMed

    Stein, Lauren M; Yosten, Gina L C; Samson, Willis K

    2016-03-15

    Adropin, a recently described peptide hormone produced in the brain and liver, has been reported to have physiologically relevant actions on glucose homeostasis and lipogenesis, and to exert significant effect on endothelial function. We describe a central nervous system action of adropin to inhibit water drinking and identify a potential adropin receptor, the orphan G protein-coupled receptor, GPR19. Reduction in GPR19 mRNA levels in medial basal hypothalamus of male rats resulted in the loss of the inhibitory effect of adropin on water deprivation-induced thirst. The identification of a novel brain action of adropin and a candidate receptor for the peptide should extend and accelerate the study of the potential therapeutic value of adropin or its mimetics for the treatment of metabolic disorders. PMID:26739651

  3. [G-protein coupled receptors. Nobel Prize 2012 for chemistry to Robert J. Lefkowitz and Brian Kobilka].

    PubMed

    Bockaert, Joël

    2012-12-01

    The 2012 Nobel Prize for chemistry has been won by Robert J. Lefkowitz and Brian Kobilka for their work on G protein-coupled receptors (GPCRs). Those receptors (3% of human genome) evolutionary are derived from one 1 or 2 ancestors and are able to recognize external message as different as light, odorants, gustative molecules and intercellular messages such as hormones and neurotransmitters. They are targets of 30-40% of therapeutic drugs. Robert J. Lefkowitz has been one of the leaders of the field from more than 40 years and has built several key concepts of the domain. Brian Kobilka was successful, in 2007, in producing a crystal structure of the β2-adrenergic receptor. This paved the way for the production of a series of almost 50 GPCR crystal structures both in inactive and active forms.

  4. Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.

    PubMed

    Burg, John S; Ingram, Jessica R; Venkatakrishnan, A J; Jude, Kevin M; Dukkipati, Abhiram; Feinberg, Evan N; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O; Ploegh, Hidde L; Garcia, K Christopher

    2015-03-01

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state. PMID:25745166

  5. Receptor-Drug Interaction: Europium Employment for Studying the Biochemical Pathway of G-Protein-Coupled Receptor Activation

    PubMed Central

    Antonio, Colabufo Nicola; Grazia, Perrone Maria; Marialessandra, Contino; Francesco, Berardi; Roberto, Perrone

    2007-01-01

    In medicinal chemistry field, the biochemical pathways, involved in 7-transmembrane domains G-protein coupled receptors (GPCRs) activation, are commonly studied to establish the activity of ligands towards GPCRs. The most studied steps are the measurement of activated GTP-α subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTPγS or [3H]cAMP as probes. Recently, the corresponding lanthanide labels Eu-GTP and Eu-cAMP as alternative to radiochemicals have been developed because they are highly sensitive, easy to automate, easily synthesized, they display a much longer shelf-life and they can be used in multilabel experiments. In the present review, the receptor-drug interaction by europium employment for studying the biochemical pathway of GPCR activation has been focused. Moreover, comparative studies between lanthanide label probes and the corresponding radiolabeled compounds have been carried out. PMID:18350113

  6. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    PubMed

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

  7. International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors--family B G protein-coupled receptors.

    PubMed

    Gardella, Thomas J; Vilardaga, Jean-Pierre

    2015-01-01

    The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

  8. Importance of the gamma-aminobutyric acid(B) receptor C-termini for G-protein coupling.

    PubMed

    Grünewald, Sylvia; Schupp, Bettina J; Ikeda, Stephen R; Kuner, Rohini; Steigerwald, Frank; Kornau, Hans-Christian; Köhr, Georg

    2002-05-01

    Functional gamma-aminobutyric acid(B) (GABA(B)) receptors assemble from two subunits, GABA(B(1)) and GABA(B(2).) This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABA(B(1)) and GABA(B(2)) for G-protein coupling. We generated a series of C-terminal mutants of GABA(B(1)) and GABA(B(2)) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABA(B(1)) functioned only in the context of specific amino acids, which constitute part of the GABA(B(1)) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABA(B(2).) In HEK293 cells, we observed that the two well-known GABA(B) receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABA(B(1)) and GABA(B(2)) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABA(B) receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABA(B) receptor may determine its

  9. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  10. G Protein-Coupled Receptor Trafficking and Signalling in the Enteric Nervous System: The Past, Present and Future.

    PubMed

    Poole, Daniel P; Bunnett, Nigel W

    2016-01-01

    G protein-coupled receptors (GPCRs) enable cells to detect and respond to changes in their extracellular environment. With over 800 members, the GPCR family includes receptors for a diverse range of agonists including olfactants, neurotransmitters and hormones. Importantly, GPCRs represent a major therapeutic target, with approximately 50 % of all current drugs acting at some aspect of GPCR signalling (Audet and Bouvier 2008). GPCRs are widely expressed by all cell types in the gastrointestinal (GI) tract and are major regulators of every aspect of gut function. Many GPCRs are internalised upon activation, and this represents one of the mechanisms through which G protein-signalling is terminated. The latency between the endocytosis of GPCRs and their recycling and resensitization is a major determinant of the cell's ability to respond to subsequent exposure to agonists. PMID:27379642

  11. Molecular mechanism for inhibition of G protein-coupled receptor kinase 2 by a selective RNA aptamer

    PubMed Central

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J. G.

    2012-01-01

    SUMMARY Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase. PMID:22727813

  12. Minireview: Role of intracellular scaffolding proteins in the regulation of endocrine G protein-coupled receptor signaling.

    PubMed

    Walther, Cornelia; Ferguson, Stephen S G

    2015-06-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies.

  13. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells★

    PubMed Central

    Engelstoft, Maja S.; Park, Won-mee; Sakata, Ichiro; Kristensen, Line V.; Husted, Anna Sofie; Osborne-Lawrence, Sherri; Piper, Paul K.; Walker, Angela K.; Pedersen, Maria H.; Nøhr, Mark K.; Pan, Jie; Sinz, Christopher J.; Carrington, Paul E.; Akiyama, Taro E.; Jones, Robert M.; Tang, Cong; Ahmed, Kashan; Offermanns, Stefan; Egerod, Kristoffer L.; Zigman, Jeffrey M.; Schwartz, Thue W.

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell. PMID:24327954

  14. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells.

    PubMed

    Engelstoft, Maja S; Park, Won-Mee; Sakata, Ichiro; Kristensen, Line V; Husted, Anna Sofie; Osborne-Lawrence, Sherri; Piper, Paul K; Walker, Angela K; Pedersen, Maria H; Nøhr, Mark K; Pan, Jie; Sinz, Christopher J; Carrington, Paul E; Akiyama, Taro E; Jones, Robert M; Tang, Cong; Ahmed, Kashan; Offermanns, Stefan; Egerod, Kristoffer L; Zigman, Jeffrey M; Schwartz, Thue W

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

  15. A biosensor-based approach toward purification and crystallization of G protein-coupled receptors.

    PubMed

    Navratilova, Iva; Pancera, Marie; Wyatt, Richard T; Myszka, David G

    2006-06-15

    Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.

  16. Optical probes based on G protein-coupled receptors - added work or added value?

    PubMed

    Stumpf, A D; Hoffmann, C

    2016-01-01

    In 2003, the first report was published that presented proof of principle for a novel class of FRET biosensors for use in living cells. This novel sensor class was built on the base of GPCRs, which represent an integral transmembrane receptor family passing the membrane seven times and are thus also called the 7TM receptor family. As an estimated number of 30% of all marketed drugs exert their effects by modulating GPCR function, these initial reports promised the gain of novel insights into receptor function. Such FRET sensors have slowly, but progressively, made their way into the standard toolbox for GPCR research as several groups are now reporting on the generation and use of these sensors. By now, FRET sensors have been reported for 18 different GPCRs, and more are expected to be added. These particular receptor sensors have been used to investigate receptor dynamics in living cells to evaluate ligand binding and ligand efficacy in real time, to study voltage and mechanosensitivity of GPCRs or to study the influence of receptor polymorphisms on receptor function in real-time. In this review we will describe the different design principles of these GPCR-based sensors and will summarize their current biological applications in living cells.

  17. Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

    NASA Astrophysics Data System (ADS)

    Märki, Iwan; Leutenegger, Marcel; Geissbuehler, Matthias; Robelek, Rudolf; Sinner, Eva-Kathrin; Lasser, Theo

    2008-02-01

    Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02μm -2min -1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

  18. Protein-based virtual screening of chemical databases. II. Are homology models of G-Protein Coupled Receptors suitable targets?

    PubMed

    Bissantz, Caterina; Bernard, Philippe; Hibert, Marcel; Rognan, Didier

    2003-01-01

    The aim of the current study is to investigate whether homology models of G-Protein-Coupled Receptors (GPCRs) that are based on bovine rhodopsin are reliable enough to be used for virtual screening of chemical databases. Starting from the recently described 2.8 A-resolution X-ray structure of bovine rhodopsin, homology models of an "antagonist-bound" form of three human GPCRs (dopamine D3 receptor, muscarinic M1 receptor, vasopressin V1a receptor) were constructed. The homology models were used to screen three-dimensional databases using three different docking programs (Dock, FlexX, Gold) in combination with seven scoring functions (ChemScore, Dock, FlexX, Fresno, Gold, Pmf, Score). Rhodopsin-based homology models turned out to be suitable, indeed, for virtual screening since known antagonists seeded in the test databases could be distinguished from randomly chosen molecules. However, such models are not accurate enough for retrieving known agonists. To generate receptor models better suited for agonist screening, we developed a new knowledge- and pharmacophore-based modeling procedure that might partly simulate the conformational changes occurring in the active site during receptor activation. Receptor coordinates generated by this new procedure are now suitable for agonist screening. We thus propose two alternative strategies for the virtual screening of GPCR ligands, relying on a different set of receptor coordinates (antagonist-bound and agonist-bound states).

  19. G-protein-coupled receptor heteromers as key players in the molecular architecture of the central nervous system.

    PubMed

    Brugarolas, Marc; Navarro, Gemma; Martínez-Pinilla, Eva; Angelats, Edgar; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2014-08-01

    The overall architecture of the nervous system, especially the CNS, is remarkable. The anatomy of the nervous system is constituted not only by macroscopic and microscopy identifiable regions and neuronal cell types, but also by protein complexes whose identification and localization require sophisticated techniques. G-protein-coupled receptors (GPCRs) constitute an example of proteins that are the key factors in the framework needed to sustain brain and nerve structure and function. The versatility underlying nervous system anatomy takes advantage of a recently discovered feature of GPCRs, the possibility to form heteromers that, placed at specific neuronal subsets and at specific locations (pre-, post-, or peri-synaptic), contribute to attain unique neural functions. PMID:24809909

  20. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking.

    PubMed

    Kang, Dong Soo; Tian, Xufan; Benovic, Jeffrey L

    2014-04-01

    The arrestin clan can now be broadly divided into three structurally similar subgroups: the originally identified arrestins (visual and β-arrestins), the α-arrestins and a group of Vps26-related proteins. The visual and β-arrestins selectively bind to agonist-occupied phosphorylated G protein-coupled receptors (GPCRs) and inhibit GPCR coupling to heterotrimeric G proteins while the β-arrestins also function as adaptor proteins to regulate GPCR trafficking and G protein-independent signaling. The α-arrestins have also recently been implicated in regulating GPCR trafficking while Vps26 regulates retrograde trafficking. In this review, we provide an overview of the α-arrestins and β-arrestins with a focus on our current understanding of how these adaptor proteins regulate GPCR trafficking.

  1. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis.

    PubMed

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W; Lira, Sergio A; Levine, Beth

    2016-03-15

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.

  2. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis.

    PubMed

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W; Lira, Sergio A; Levine, Beth

    2016-03-15

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling. PMID:26929373

  3. G-protein Coupled Receptor Signaling in Pluripotent Stem Cell-derived Cardiovascular Cells: Implications for Disease Modeling

    PubMed Central

    Dolatshad, Nazanin F.; Hellen, Nicola; Jabbour, Richard J.; Harding, Sian E.; Földes, Gabor

    2015-01-01

    Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies. PMID:26697426

  4. Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour

    PubMed Central

    Doi, Masao; Murai, Iori; Kunisue, Sumihiro; Setsu, Genzui; Uchio, Naohiro; Tanaka, Rina; Kobayashi, Sakurako; Shimatani, Hiroyuki; Hayashi, Hida; Chao, Hsu-Wen; Nakagawa, Yuuki; Takahashi, Yukari; Hotta, Yunhong; Yasunaga, Jun-ichirou; Matsuoka, Masao; Hastings, Michael H.; Kiyonari, Hiroshi; Okamura, Hitoshi

    2016-01-01

    G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz. PMID:26882873

  5. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

    PubMed

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  6. Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases*♦

    PubMed Central

    Komolov, Konstantin E.; Bhardwaj, Anshul; Benovic, Jeffrey L.

    2015-01-01

    G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs. PMID:26032409

  7. Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

    PubMed

    Homan, Kristoff T; Waldschmidt, Helen V; Glukhova, Alisa; Cannavo, Alessandro; Song, Jianliang; Cheung, Joseph Y; Koch, Walter J; Larsen, Scott D; Tesmer, John J G

    2015-08-21

    G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

  8. Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor.

    PubMed

    Offertáler, László; Mo, Fong-Ming; Bátkai, Sándor; Liu, Jie; Begg, Malcolm; Razdan, Raj K; Martin, Billy R; Bukoski, Richard D; Kunos, George

    2003-03-01

    The cannabinoid analog abnormal cannabidiol [abn-cbd; (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to CB(1) or CB(2) receptors, yet it acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasorelaxation by abn-cbd is endothelium-dependent, pertussis toxin-sensitive, and is inhibited by the BK(Ca) channel inhibitor charybdotoxin, but not by the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester or by the vanilloid VR1 receptor antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB(1) or CB(2) receptors and does not cause vasorelaxation at concentrations up to 30 microM, but it does cause concentration-dependent (1-30 microM) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd but not the hypotensive effect of the CB(1) receptor agonist (-)-11-OH-Delta(9)-tetrahydrocannabinol dimethylheptyl. In human umbilical vein endothelial cells, abn-cbd induces phosphorylation of p42/44 mitogen-activated protein kinase and protein kinase B/Akt, which is inhibited by O-1918, by pertussis toxin or by phosphatidylinositol 3 (PI3) kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and that O-1918 is a selective, silent antagonist of an endothelial "anandamide receptor", which is distinct from CB(1) or CB(2) receptors and is coupled through G(i)/G(o) to the PI3 kinase/Akt signaling pathway.

  9. A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution

    PubMed Central

    Klenk, Christoph; Ehrenmann, Janosch; Schütz, Marco; Plückthun, Andreas

    2016-01-01

    Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded receptor, are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis. PMID:26887595

  10. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    SciTech Connect

    Cline, Gary W.; Zhao, Xiaojian; Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L.

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  11. Tick G protein-coupled receptors as targets for development of new acaricides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The GPCR class of receptors is a source of many pharmacologicals in human medicine and are still being pursued in research programs as promising targets for drug development. GPCRs have been identified in arthropods and with the advent of expressed sequence tag (EST) and genome projects in the ticks...

  12. Do G protein-coupled receptors expressed in human lingual epithelium interact with HPV11?

    PubMed

    Durzyński, Lukasz; Gaudin, Jean-Charles; Breuils, Laure; Szydłowski, Jaroslaw; Goździcka-Józefiak, Anna; Haertlé, Thomas

    2007-10-01

    Human papillomaviruses infect epithelia but little is known about the nature of cell surface receptors interacting with the viral particles. It has been proposed that glycosaminoglycans and integrins may be involved in the attachment process. In the present study, the putative interactions of virus-like particles of human papillomavirus type 11 (HPV11), which present a tropism for nasopharyngeal epithelia, with olfactory and taste receptors expressed in the human lingual epithelium were studied. The L1 protein of HPV11 was produced in insect cells. The presence of L1 virus-like particles was analyzed by ELISA using monoclonal antibodies specific for full-size particles and by electron microscopy. Using immunofluorescence, it was observed that virus-like particles interacted with taste buds from murine tongue, with the tagged human olfactory receptor hJCG5 expressed in HEK-293 but not with the tagged taste receptor hT2R4. This therefore suggests that hJCG5 may be involved in the adsorption process of HPV11 to lingual epithelium serving as a so-called "adsorption-adhesive molecule." PMID:17705193

  13. Sex peptides and MIPs can activate the same G protein-coupled receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In many animal species, copulation elicits a number of physiological and behavioral changes in the female partner. In Drosophila melanogaster, the main molecular effector of these physiological responses has been identified as sex peptide (SP). The sex peptide receptor (SPR) has been characterized a...

  14. Identification and comparative analysis of G protein-coupled receptors in Pediculus humanus humanus.

    PubMed

    Li, Chengjun; Song, Xiaowen; Chen, Xuhong; Liu, Xing; Sang, Ming; Wu, Wei; Yun, Xiaopei; Hu, Xingxing; Li, Bin

    2014-07-01

    The body louse has the smallest genome size among the known genome-sequenced insects. Here, 81 GPCRs were identified in Pediculus humanus humanus, 56, 14, 6 and 5 GPCRs for family-A, -B, -C and -D, respectively. These GPCRs constitute the comparable repertoire of GPCRs with other insects. Moreover, it contains a more complete set of neuropeptide and protein hormone receptors not even than Acyrthosiphon pisum but also Drosophila melanogaster, for example, Sulfakinin, Corazonin, Trissin and PTHRL only presented in P. h. humanus but lost either in A. pisum or D. melanogaster. However, it has less duplication among the sub-families. Meanwhile, ACP, AVPL, HE6 receptors and Boss were also absent from P. h. humanus. These results indicated that the repertoire of GPCRs in P. h. humanus were not affected by its smallest genome size, and further suggested that P. h. humanus has a relatively original and concise GPCR regulation system. PMID:24952173

  15. EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors.

    PubMed

    Bojjireddy, Naveen; Guzman-Hernandez, Maria Luisa; Reinhard, Nathalie Renée; Jovic, Marko; Balla, Tamas

    2015-01-01

    The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes.

  16. EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors

    PubMed Central

    Bojjireddy, Naveen; Guzman-Hernandez, Maria Luisa; Reinhard, Nathalie Renée; Jovic, Marko; Balla, Tamas

    2015-01-01

    ABSTRACT The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca2+ response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes. PMID:25380825

  17. Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors.

    PubMed

    Binet, Virginie; Duthey, Béatrice; Lecaillon, Jennifer; Vol, Claire; Quoyer, Julie; Labesse, Gilles; Pin, Jean-Philippe; Prézeau, Laurent

    2007-04-20

    G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.

  18. Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids

    PubMed Central

    2015-01-01

    We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide–alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism. PMID:25524496

  19. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    SciTech Connect

    Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  20. Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

    PubMed Central

    Shivnaraine, Rabindra V; Kelly, Brendan; Sankar, Krishana S; Redka, Dar'ya S; Han, Yi Rang; Huang, Fei; Elmslie, Gwendolynne; Pinto, Daniel; Li, Yuchong; Rocheleau, Jonathan V; Gradinaru, Claudiu C; Ellis, John; Wells, James W

    2016-01-01

    The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations. DOI: http://dx.doi.org/10.7554/eLife.11685.001 PMID:27151542

  1. Synergistically acting agonists and antagonists of G protein-coupled receptors prevent photoreceptor cell degeneration.

    PubMed

    Chen, Yu; Palczewska, Grazyna; Masuho, Ikuo; Gao, Songqi; Jin, Hui; Dong, Zhiqian; Gieser, Linn; Brooks, Matthew J; Kiser, Philip D; Kern, Timothy S; Martemyanov, Kirill A; Swaroop, Anand; Palczewski, Krzysztof

    2016-01-01

    Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration-approved drugs that act on different G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders. PMID:27460988

  2. The ligand specificity of the G-protein-coupled receptor GPR34.

    PubMed

    Ritscher, Lars; Engemaier, Eva; Stäubert, Claudia; Liebscher, Ines; Schmidt, Philipp; Hermsdorf, Thomas; Römpler, Holger; Schulz, Angela; Schöneberg, Torsten

    2012-05-01

    Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities. PMID:22348703

  3. Smoothened Signaling in Vertebrates Is Facilitated by a G Protein-coupled Receptor Kinase

    PubMed Central

    Philipp, Melanie; Fralish, Gregory B.; Meloni, Alison R.; Chen, Wei; MacInnes, Alyson W.; Barak, Lawrence S.

    2008-01-01

    Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein–coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein–dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development. PMID:18815277

  4. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.

    PubMed

    Ye, Shixin; Köhrer, Caroline; Huber, Thomas; Kazmi, Manija; Sachdev, Pallavi; Yan, Elsa C Y; Bhagat, Aditi; RajBhandary, Uttam L; Sakmar, Thomas P

    2008-01-18

    G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors. PMID:17993461

  5. Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand

    PubMed Central

    2015-01-01

    The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design—the photoswitchable orthogonal remotely tethered ligand (PORTL)—that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized. PMID:27162996

  6. Absorption of PCB126 by upper airways impairs G protein-coupled receptor-mediated immune response

    NASA Astrophysics Data System (ADS)

    Shimada, Ana Lúcia B.; Cruz, Wesley S.; Loiola, Rodrigo A.; Drewes, Carine C.; Dörr, Fabiane; Figueiredo, Natália G.; Pinto, Ernani; Farsky, Sandra H. P.

    2015-10-01

    PCB126 is a dioxin-like polychlorinated biphenyl (PCB) environmental pollutant with a significant impact on human health, as it bioaccumulates and causes severe toxicity. PCB126-induced immune toxicity has been described, although the mechanisms have not been fully elucidated. In this study, an in vivo protocol of PCB126 intoxication into male Wistar rats by intranasal route was used, which has not yet been described. The intoxication was characterised by PCB126 accumulation in the lungs and liver, and enhanced aryl hydrocarbon receptor expression in the liver, lungs, kidneys, and adipose tissues. Moreover, an innate immune deficiency was characterised by impairment of adhesion receptors on blood leukocytes and by reduced blood neutrophil locomotion and oxidative burst activation elicited by ex vivo G protein-coupled receptor (GPCR) activation. Specificity of PCB126 actions on the GPCR pathway was shown by normal burst oxidative activation evoked by Toll-like receptor 4 and protein kinase C direct activation. Moreover, in vivo PCB180 intoxication did not alter adhesion receptors on blood leukocytes either blood neutrophil locomotion, and only partially reduced the GPCR-induced burst oxidative activation on neutrophils. Therefore, a novel mechanism of in vivo PCB126 toxicity is described which impairs a pivotal inflammatory pathway to the host defence against infections.

  7. International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, Classification, and Pharmacology of G Protein-Coupled Melatonin Receptors

    PubMed Central

    Delagrange, Philippe; Krause, Diana N.; Sugden, David; Cardinali, Daniel P.; Olcese, James

    2010-01-01

    The hormone melatonin (5-methoxy-N-acetyltryptamine) is synthesized primarily in the pineal gland and retina, and in several peripheral tissues and organs. In the circulation, the concentration of melatonin follows a circadian rhythm, with high levels at night providing timing cues to target tissues endowed with melatonin receptors. Melatonin receptors receive and translate melatonin's message to influence daily and seasonal rhythms of physiology and behavior. The melatonin message is translated through activation of two G protein-coupled receptors, MT1 and MT2, that are potential therapeutic targets in disorders ranging from insomnia and circadian sleep disorders to depression, cardiovascular diseases, and cancer. This review summarizes the steps taken since melatonin's discovery by Aaron Lerner in 1958 to functionally characterize, clone, and localize receptors in mammalian tissues. The pharmacological and molecular properties of the receptors are described as well as current efforts to discover and develop ligands for treatment of a number of illnesses, including sleep disorders, depression, and cancer. PMID:20605968

  8. Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

    PubMed Central

    Miao, Yinglong; Caliman, Alisha D.; McCammon, J. Andrew

    2015-01-01

    G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding. PMID:25863070

  9. Expression of G protein-coupled receptor 56 is associated with tumor progression in non-small-cell lung carcinoma patients

    PubMed Central

    Song, Yanjie; Li, Aiqin; Zhang, Li; Duan, Lingling

    2016-01-01

    Background G protein-coupled receptor 56 (GPR56) is an adhesion G protein-coupled receptor with essential functions for cell physiology and survival, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of GPR56 expression with clinicopathological parameters and prognosis in non-small-cell lung carcinoma (NSCLC). Methods The levels of GPR56 were evaluated by immunohistochemistry in 157 NSCLC tissue samples. The association between GPR56 and clinicopathological parameters was evaluated by χ2 test. Univariate and multivariate analyses were performed to demonstrate the prognosis role of GPR56. The function of GPR56 in NSCLC cell lines was also explored through overexpression and knockdown studies. Results The expression level of GPR56 in tumor tissues was significantly correlated with the TNM stage of NSCLC (P=0.005). Univariate and multivariate analyses revealed that GPR56 can act as an independent prognostic factor for overall survival. Furthermore, through overexpression and knockdown experiments, we confirmed that GPR56 can promote the proliferation and invasion of NSCLC cells. Conclusion GPR56 plays an important role in tumor development and may serve as a promising target for prognostic prediction in NSCLC. PMID:27462165

  10. G Protein-Coupled Receptors - Targets for Fragment-based Drug Discovery.

    PubMed

    Lawson, Alastair D G

    2015-01-01

    As the considerable technical challenges involved with generating crystal structures of G (guanine nucleotide- binding) protein-coupled receptors (GPCRs) are starting to be successfully addressed, opportunities to apply fragment-based drug discovery (FBDD) to this class of target are becoming a reality. GPCRs represent a large and important family of drug targets with considerable clinical and commercial interest. While their general seven transmembrane helix bundle structures are amenable to therapeutic intervention with small molecules, to date successful drugs have primarily been discovered using traditional competitive or function-based screening. With advances in biophysical screening techniques such as Surface Plasmon Resonance (SPR) and Target-Immobilised NMR Screening (TINS), being matched to developments in molecular dynamics simulations, virtual screening and stabilisation of biologically relevant conformations of GPCRs, structure-based approaches using fragment starting points are beginning to be applied to the discovery of new generations of small molecules.

  11. G Protein-Coupled Receptors - Targets for Fragment-based Drug Discovery.

    PubMed

    Lawson, Alastair D G

    2015-01-01

    As the considerable technical challenges involved with generating crystal structures of G (guanine nucleotide- binding) protein-coupled receptors (GPCRs) are starting to be successfully addressed, opportunities to apply fragment-based drug discovery (FBDD) to this class of target are becoming a reality. GPCRs represent a large and important family of drug targets with considerable clinical and commercial interest. While their general seven transmembrane helix bundle structures are amenable to therapeutic intervention with small molecules, to date successful drugs have primarily been discovered using traditional competitive or function-based screening. With advances in biophysical screening techniques such as Surface Plasmon Resonance (SPR) and Target-Immobilised NMR Screening (TINS), being matched to developments in molecular dynamics simulations, virtual screening and stabilisation of biologically relevant conformations of GPCRs, structure-based approaches using fragment starting points are beginning to be applied to the discovery of new generations of small molecules. PMID:26126904

  12. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

    PubMed Central

    Moore, Amanda R; Ceraudo, Emilie; Sher, Jessica J; Guan, Youxin; Shoushtari, Alexander N; Chang, Matthew T; Zhang, Jenny Q; Walczak, Edward G; Kazmi, Manija A; Taylor, Barry S; Huber, Thomas; Chi, Ping; Sakmar, Thomas P; Chen, Yu

    2016-01-01

    Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling 1–3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis. PMID:27089179

  13. Altered immune response in mice deficient for the G protein-coupled receptor GPR34.

    PubMed

    Liebscher, Ines; Müller, Uwe; Teupser, Daniel; Engemaier, Eva; Engel, Kathrin M Y; Ritscher, Lars; Thor, Doreen; Sangkuhl, Katrin; Ricken, Albert; Wurm, Antje; Piehler, Daniel; Schmutzler, Sandra; Fuhrmann, Herbert; Albert, Frank W; Reichenbach, Andreas; Thiery, Joachim; Schöneberg, Torsten; Schulz, Angela

    2011-01-21

    The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges. PMID:21097509

  14. Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34*

    PubMed Central

    Liebscher, Ines; Müller, Uwe; Teupser, Daniel; Engemaier, Eva; Engel, Kathrin M. Y.; Ritscher, Lars; Thor, Doreen; Sangkuhl, Katrin; Ricken, Albert; Wurm, Antje; Piehler, Daniel; Schmutzler, Sandra; Fuhrmann, Herbert; Albert, Frank W.; Reichenbach, Andreas; Thiery, Joachim; Schöneberg, Torsten; Schulz, Angela

    2011-01-01

    The X-chromosomal GPR34 gene encodes an orphan Gi protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges. PMID:21097509

  15. Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell.

    PubMed

    Wang, D; Surratt, C K; Sadée, W

    2000-08-01

    Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist-stimulated G protein coupling of the mu-opioid receptor (OP(3)) through direct interaction with the third intracellular (i3) loop of the receptor. Here we have investigated the role of CaM in regulating changes in OP(3)-G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist-stimulated G protein coupling by OP(3) was measured before and after morphine pretreatment by incorporation of guanosine 5'-O-(3-[(35)S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP(3) cDNA. The opioid antagonist beta-chlornaltrexamine fully suppressed basal G protein coupling of OP(3), providing a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, agonist-stimulated coupling was diminished (desensitization), resulting in a substantially flattened morphine dose-response curve. To test whether CaM is involved in these changes, we constructed OP(3)-i3 loop mutants with reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP(3) receptors was higher than that of the wild-type receptor and, moreover, unaffected by morphine pretreatment, whereas desensitization to agonist stimulation was only slightly attenuated. Therefore, CaM-OP(3) interactions appear to play only a minor role in the desensitization of OP(3). In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP(3), thereby resolving the paradox that OP(3) displays both desensitization and sensitization during morphine treatment.

  16. Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

    PubMed Central

    Daumas, Frédéric; Destainville, Nicolas; Millot, Claire; Lopez, André; Dean, David; Salomé, Laurence

    2003-01-01

    Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the μ-opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients. PMID:12524289

  17. Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes.

    PubMed

    Graaf, Chris de; Donnelly, Dan; Wootten, Denise; Lau, Jesper; Sexton, Patrick M; Miller, Laurence J; Ahn, Jung-Mo; Liao, Jiayu; Fletcher, Madeleine M; Yang, Dehua; Brown, Alastair J H; Zhou, Caihong; Deng, Jiejie; Wang, Ming-Wei

    2016-10-01

    The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders. PMID:27630114

  18. Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens.

    PubMed

    Tanaka, Yoshiaki; Suetsugu, Yoshitaka; Yamamoto, Kimiko; Noda, Hiroaki; Shinoda, Tetsuro

    2014-03-01

    The genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors were identified in the brown planthopper (BPH), Nilaparvata lugens (Stål) by transcriptome analysis (RNA-seq). Forty-eight candidate genes were found to encode neuropeptides or peptide hormones. These include all known insect neuropeptides and neurohormones, with the exception of neuropeptide-like precursor 2 (NPLP2) and trissin. The gene coding for prothoracicotropic hormone (PTTH) was first identified from hemimetabolous insect. A total of 57 putative neuropeptide GPCR genes were identified and phylogenetic analysis showed most of them to be closely related to insect GPCRs. A notable finding was the occurrence of vertebrate hormone receptors, thyrotropin-releasing hormone receptor (TRHR)-like GPCR and parathyroid hormone receptor (PTHR)-like GPCRs. These results suggest that N. lugens possesses the most comprehensive neuropeptide system yet found in insects. Moreover, our findings demonstrate the power of RNA-seq as a tool for analyzing the neuropeptide-related genes in the absence of whole genome sequence information.

  19. Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.

    PubMed

    Jonas, Kim C; Fanelli, Francesca; Huhtaniemi, Ilpo T; Hanyaloglu, Aylin C

    2015-02-13

    Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼ 8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHR(B-)) and a signaling-deficient (LHR(S-)) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHR(B-) to LHR(S-) modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

  20. The role of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

    PubMed

    Sawamura, Ryo; Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

    2015-03-01

    Clarification of the mechanism of chickens' taste sense will provide meaningful information for creating and improving new feedstuff for chickens, because the character of taste receptors in oral tissues affects feeding behavior in animals. Although fatty acids are partly recognized via G-protein coupled receptor 120 (GPR120) for fat taste in mammalian oral tissues, the fat taste receptor of chickens has not been elucidated. Here we cloned chicken GPR120 (cGPR120) from the chicken palate, which contains taste buds. By using Ca(2+) imaging methods, we identified oleic acid and linoleic acid as cGPR120 agonists. Interestingly, in a behavioral study the chickens preferred corn oil-rich feed over mineral oil (control oil)-rich feed. Because corn oil contains high amounts of oleic acid and linoleic acid, this result was thought to be reasonable. Taken together, the present results suggest that cGPR120 is one of the functional fat taste receptors in chickens.

  1. A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors.

    PubMed

    Duvernay, Matthew T; Dong, Chunmin; Zhang, Xiaoping; Robitaille, Mélanie; Hébert, Terence E; Wu, Guangyu

    2009-05-01

    The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.

  2. The GPS Motif Is a Molecular Switch for Bimodal Activities of Adhesion Class G Protein-Coupled Receptors

    PubMed Central

    Prömel, Simone; Frickenhaus, Marie; Hughes, Samantha; Mestek, Lamia; Staunton, David; Woollard, Alison; Vakonakis, Ioannis; Schöneberg, Torsten; Schnabel, Ralf; Russ, Andreas P.; Langenhan, Tobias

    2012-01-01

    Summary Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions. PMID:22938866

  3. Divergent β-arrestin-dependent signaling events are dependent upon sequences within G-protein-coupled receptor C termini.

    PubMed

    Pal, Kasturi; Mathur, Maneesh; Kumar, Puneet; DeFea, Kathryn

    2013-02-01

    β-Arrestins are multifunctional adaptor proteins that, upon recruitment to an activated G-protein-coupled receptor, can promote desensitization of G-protein signaling and receptor internalization while simultaneously eliciting an independent signal. The result of β-arrestin signaling depends upon the activating receptor. For example, activation of two Gα(q)-coupled receptors, protease-activated receptor-2 (PAR(2)) and neurokinin-1 receptor (NK1R), results in drastically different signaling events. PAR(2) promotes β-arrestin-dependent membrane-sequestered extracellular signal-regulated kinase (ERK1/2) activation, cofilin activation, and cell migration, whereas NK1R promotes nuclear ERK1/2 activation and proliferation. Using bioluminescence resonance energy transfer to monitor receptor/β-arrestin interactions in real time, we observe that PAR(2) has a higher apparent affinity for both β-arrestins than does NK1R, recruits them at a faster rate, and exhibits more rapid desensitization of the G-protein signal. Furthermore, recruitment of β-arrestins to PAR(2) does not require prior Gα(q) signaling events, whereas inhibition of Gα(q) signaling intermediates inhibits recruitment of β-arrestins to NK1R. Using chimeric receptors in which the C terminus of PAR(2) is fused to the N terminus of NK1R and vice versa and a critical Ser/Thr mutant of PAR(2), we demonstrate that interactions between β-arrestins and specific phosphoresidues in the C termini of each receptor are crucial for determining the rate and magnitude of β-arrestin recruitment as well as the ultimate signaling outcome.

  4. Serotonin Signaling in Schistosoma mansoni: A Serotonin–Activated G Protein-Coupled Receptor Controls Parasite Movement

    PubMed Central

    Rashid, Mohammed; Ribeiro, Paula

    2014-01-01

    Serotonin is an important neuroactive substance in all the parasitic helminths. In Schistosoma mansoni, serotonin is strongly myoexcitatory; it potentiates contraction of the body wall muscles and stimulates motor activity. This is considered to be a critical mechanism of motor control in the parasite, but the mode of action of serotonin is poorly understood. Here we provide the first molecular evidence of a functional serotonin receptor (Sm5HTR) in S. mansoni. The schistosome receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to serotonergic type 7 (5HT7) receptors from other species. Functional expression studies in transfected HEK 293 cells showed that Sm5HTR is a specific serotonin receptor and it signals through an increase in intracellular cAMP, consistent with a 5HT7 signaling mechanism. Immunolocalization studies with a specific anti-Sm5HTR antibody revealed that the receptor is abundantly distributed in the worm's nervous system, including the cerebral ganglia and main nerve cords of the central nervous system and the peripheral innervation of the body wall muscles and tegument. RNA interference (RNAi) was performed both in schistosomulae and adult worms to test whether the receptor is required for parasite motility. The RNAi-suppressed adults and larvae were markedly hypoactive compared to the corresponding controls and they were also resistant to exogenous serotonin treatment. These results show that Sm5HTR is at least one of the receptors responsible for the motor effects of serotonin in S. mansoni. The fact that Sm5HTR is expressed in nerve tissue further suggests that serotonin stimulates movement via this receptor by modulating neuronal output to the musculature. Together, the evidence identifies Sm5HTR as an important neuronal protein and a key component of the motor control apparatus in S. mansoni. PMID:24453972

  5. Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease

    PubMed Central

    Leo, Vincenzo C.; Sabi, Essa; Cunningham, Margaret R.; Eikenboom, Jeroen C.; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C.; Watson, Steve P.; Mundell, Stuart J.; Daly, Martina E.

    2015-01-01

    The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder. PMID:26630678

  6. Identification of a Chemoattractant G-Protein-Coupled Receptor for Folic Acid that Controls Both Chemotaxis and Phagocytosis.

    PubMed

    Pan, Miao; Xu, Xuehua; Chen, Yong; Jin, Tian

    2016-02-22

    Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. The social amoeba Dictyostelium discoideum is a professional phagocyte that chases bacteria through chemotaxis and engulfs them as food via phagocytosis. G-protein-coupled receptors (GPCRs) are known for detecting chemoattractants and directing cell migration, but their roles in phagocytosis are not clear. Here, we developed a quantitative phosphoproteomic technique to discover signaling components. Using this approach, we discovered the long sought after folic acid receptor, fAR1, in D. discoideum. We showed that the seven-transmembrane receptor fAR1 is required for folic acid-mediated signaling events. Significantly, we discovered that fAR1 is essential for both chemotaxis and phagocytosis of bacteria, thereby representing a chemoattractant GPCR that mediates not only chasing but also ingesting bacteria. We revealed that a phagocyte is able to internalize particles via a chemoattractant-mediated engulfment process. We propose that mammalian phagocytes may also use this mechanism to engulf and ingest bacterial pathogens.

  7. Digestive physiology of the pig symposium: G protein-coupled receptors in nutrient chemosensation and gastrointestinal hormone secretion.

    PubMed

    Liou, A P

    2013-05-01

    The gastrointestinal tract is a highly effective and efficient organ system that digests and absorbs nutrients, contributes to the regulation of glucose homeostasis, and signals postprandial satiety. A network of enteroendocrine cells orchestrates these events through the release of neuropeptide hormones secreted in response to the specific nutrient components within the intraluminal milieu. Nutrient chemosensing by these cells is mediated by cell membrane proteins that have been localized to hormone-producing cells. However, functional studies of the nutrient detection abilities of the endocrine cell population have been limited due to its rare and singly distributed cell type. Recent technological advances have enabled investigations with primary endocrine cells that promise to enhance our current understanding of enteroendocrine cell biology. This review focuses on a particular subset of chemosensing receptors, the G protein-coupled receptors (GPCR), that have been identified as putative nutrient sensors of the major macronutrients, lipids, proteins, and carbohydrates by enteroendocrine cells. The contributions of these receptors in directly activating and stimulating hormone secretion in several subsets of enteroendocrine cells will be discussed, based on evidence gathered by functional studies in animal models, in vitro studies in endocrine cell lines, and newly described findings in primary endocrine cells. Key insights in chemosensory detection and hormone secretion from enteroendocrine cells may help further the studies in larger animal models and guide the formulation of feed or supplements to influence the gastrointestinal signals regulating optimal food intake, absorptive capacity, and growth. PMID:23230119

  8. Identification of a Chemoattractant G-Protein-Coupled Receptor for Folic Acid that Controls Both Chemotaxis and Phagocytosis.

    PubMed

    Pan, Miao; Xu, Xuehua; Chen, Yong; Jin, Tian

    2016-02-22

    Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. The social amoeba Dictyostelium discoideum is a professional phagocyte that chases bacteria through chemotaxis and engulfs them as food via phagocytosis. G-protein-coupled receptors (GPCRs) are known for detecting chemoattractants and directing cell migration, but their roles in phagocytosis are not clear. Here, we developed a quantitative phosphoproteomic technique to discover signaling components. Using this approach, we discovered the long sought after folic acid receptor, fAR1, in D. discoideum. We showed that the seven-transmembrane receptor fAR1 is required for folic acid-mediated signaling events. Significantly, we discovered that fAR1 is essential for both chemotaxis and phagocytosis of bacteria, thereby representing a chemoattractant GPCR that mediates not only chasing but also ingesting bacteria. We revealed that a phagocyte is able to internalize particles via a chemoattractant-mediated engulfment process. We propose that mammalian phagocytes may also use this mechanism to engulf and ingest bacterial pathogens. PMID:26906738

  9. The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting.

    PubMed

    Dores, Michael R; Lin, Huilan; J Grimsey, Neil; Mendez, Francisco; Trejo, JoAnn

    2015-12-15

    The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

  10. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.

    PubMed

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  11. G protein coupled receptor signaled apoptosis is associated with activation of a cation insensitive acidic endonuclease and intracellular acidification.

    PubMed

    Sharma, K; Srikant, C B

    1998-01-01

    Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.

  12. Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

    PubMed Central

    Cannavo, Alessandro; Liccardo, Daniela; Eguchi, Akito; Elliott, Katherine J.; Traynham, Christopher J.; Ibetti, Jessica; Eguchi, Satoru; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe; Koch, Walter J.

    2016-01-01

    Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels. PMID:26932512

  13. High-throughput screening of interactions between G protein-coupled receptors and ligands using confocal optics microscopy.

    PubMed

    Zemanová, Lenka; Schenk, Andreas; Valler, Martin J; Nienhaus, G Ulrich; Heilker, Ralf

    2005-01-01

    Interactions of extracellular ligands with proteins in the cellular plasma membrane are the starting point for various intracellular signaling cascades. In the pharmaceutical industry, particular attention has been paid to G protein- coupled receptors (GPCRs), which are involved in various disease processes. In so-called high-throughput screening (HTS) campaigns, large medicinal chemistry compound libraries were searched for bioactive molecules that would either induce or inhibit the activity of a specific disease-relevant GPCR. In the respective drug discovery assays, the test compound typically competes with the physiological ligand for a binding site on the receptor. The transmembrane receptor is prepared in the form of membrane fragments or, as described here, in so-called virus-like particles (VLiPs). As hundreds of thousands of test compounds must be analyzed, there is a strict need for low volume binding assays to save the expensive bioreagents, and to reduce the consumption of the test compounds. In this chapter, we describe the application of confocal optics microscopy to measure GPCR ligand interactions in low microliter assay volumes.

  14. Improving homology modeling of G-protein coupled receptors through multiple-template derived conserved inter-residue interactions.

    PubMed

    Chaudhari, Rajan; Heim, Andrew J; Li, Zhijun

    2015-05-01

    Evidenced by the three-rounds of G-protein coupled receptors (GPCR) Dock competitions, improving homology modeling methods of helical transmembrane proteins including the GPCRs, based on templates of low sequence identity, remains an eminent challenge. Current approaches addressing this challenge adopt the philosophy of "modeling first, refinement next". In the present work, we developed an alternative modeling approach through the novel application of available multiple templates. First, conserved inter-residue interactions are derived from each additional template through conservation analysis of each template-target pairwise alignment. Then, these interactions are converted into distance restraints and incorporated in the homology modeling process. This approach was applied to modeling of the human β2 adrenergic receptor using the bovin rhodopsin and the human protease-activated receptor 1 as templates and improved model quality was demonstrated compared to the homology model generated by standard single-template and multiple-template methods. This method of "refined restraints first, modeling next", provides a fast and complementary way to the current modeling approaches. It allows rational identification and implementation of additional conserved distance restraints extracted from multiple templates and/or experimental data, and has the potential to be applicable to modeling of all helical transmembrane proteins.

  15. Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

    PubMed

    Guo, Shuohan; Zhang, Xiaohan; Zheng, Mei; Zhang, Xiaowei; Min, Chengchun; Wang, Zengtao; Cheon, Seung Hoon; Oak, Min-Ho; Nah, Seung-Yeol; Kim, Kyeong-Man

    2015-10-01

    Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

  16. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

    PubMed Central

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A.

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  17. Superfamily of genes encoding G protein-coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae).

    PubMed

    Wu, S-F; Yu, H-Y; Jiang, T-T; Gao, C-F; Shen, J-L

    2015-08-01

    G protein-coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth-and-death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress-tolerance gene, Mth, in P. xylostella. The evolution by the birth-and-death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides. PMID:25824261

  18. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis

    PubMed Central

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi

    2016-01-01

    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors. PMID:27353701

  19. Functional assay for T4 lysozyme-engineered G Protein-Coupled Receptors with an ion channel reporter

    PubMed Central

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J.

    2013-01-01

    Summary: Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains in order to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that Ion Channel-Coupled Receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. PMID:24268646

  20. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.

    PubMed

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi

    2016-01-01

    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors. PMID:27353701

  1. Constitutive Activation of G Protein-Coupled Receptors and Diseases: Insights into Mechanisms of Activation and Therapeutics

    PubMed Central

    Tao, Ya-Xiong

    2008-01-01

    The existence of constitutive activity for G protein-coupled receptors (GPCRs) was first described in 1980s. In 1991, the first naturally occurring constitutively active mutations in GPCRs that cause diseases were reported in rhodopsin. Since then, numerous constitutively active mutations that cause human diseases were reported in several additional receptors. More recently, loss of constitutive activity was postulated to also cause diseases. Animal models expressing some of these mutants confirmed the roles of these mutations in the pathogenesis of the diseases. Detailed functional studies of these naturally occurring mutations, combined with homology modeling using rhodopsin crystal structure as the template, lead to important insights into the mechanism of activation in the absence of crystal structure of GPCRs in active state. Search for inverse agonists on these receptors will be critical for correcting the diseases cause by activating mutations in GPCRs. Theoretically, these inverse agonists are better therapeutics than neutral antagonists in treating genetic diseases caused by constitutively activating mutations in GPCRs. PMID:18768149

  2. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor.

    PubMed

    Miao, Yinglong; Goldfeld, Dahlia Anne; Moo, Ee Von; Sexton, Patrick M; Christopoulos, Arthur; McCammon, J Andrew; Valant, Celine

    2016-09-20

    Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies. PMID:27601651

  3. Regulation of group II metabotropic glutamate receptors by G protein-coupled receptor kinases: mGlu2 receptors are resistant to homologous desensitization.

    PubMed

    Iacovelli, L; Molinaro, G; Battaglia, G; Motolese, M; Di Menna, L; Alfiero, M; Blahos, J; Matrisciano, F; Corsi, M; Corti, C; Bruno, V; De Blasi, A; Nicoletti, F

    2009-04-01

    We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients. PMID:19164443

  4. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells.

    PubMed

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-02-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs.

  5. G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.

    PubMed

    Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie; Bräuner-Osborne, Hans

    2014-01-01

    Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.

  6. TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch.

    PubMed

    Wilson, Sarah R; Gerhold, Kristin A; Bifolck-Fisher, Amber; Liu, Qin; Patel, Kush N; Dong, Xinzhong; Bautista, Diana M

    2011-05-01

    Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. In many cases, pathological itch is insensitive to antihistamine treatment. Recent studies have identified members of the Mas-related G protein-coupled receptor (Mrgpr) family that are activated by mast cell mediators and promote histamine-independent itch. MrgprA3 and MrgprC11 act as receptors for the pruritogens chloroquine and BAM8-22, respectively. However, the signaling pathways and transduction channels activated downstream of these pruritogens are largely unknown. We found that TRPA1 is the downstream target of both MrgprA3 and MrgprC11 in cultured sensory neurons and heterologous cells. TRPA1 is required for Mrgpr-mediated signaling, as sensory neurons from TRPA1-deficient mice exhibited markedly diminished responses to chloroquine and BAM8-22. Similarly, TRPA1-deficient mice displayed little to no scratching in response to these pruritogens. Our findings indicate that TRPA1 is an essential component of the signaling pathways that promote histamine-independent itch.

  7. Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

    PubMed

    Lu, Bin; Chen, Linjie; Zhang, Yaping; Shi, Ying; Zhou, Naiming

    2016-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery. PMID:26928549

  8. Interaction of Glucagon G-Protein Coupled Receptor with Known Natural Antidiabetic Compounds: Multiscoring In Silico Approach

    PubMed Central

    Baig, M. H.; Ahmad, K.; Hasan, Q.; Khan, M. K. A.; Rao, N. S.; Kamal, M. A.; Choi, I.

    2015-01-01

    Glucagon receptor (GCGR) is a secretin-like (class B) family of G-protein coupled receptors (GPCRs) in humans that plays an important role in elevating the glucose concentration in blood and has thus become one of the promising therapeutic targets for treatment of type 2 diabetes mellitus. GCGR based inhibitors for the treatment of type 2 diabetes are either glucagon neutralizers or small molecular antagonists. Management of diabetes without any side effects is still a challenge to the medical system, and the search for a new and effective natural GCGR antagonist is an important area for the treatment of type 2 diabetes. In the present study, a number of natural compounds containing antidiabetic properties were selected from the literature and their binding potential against GCGR was determined using molecular docking and other in silico approaches. Among all selected natural compounds, curcumin was found to be the most effective compound against GCGR followed by amorfrutin 1 and 4-hydroxyderricin. These compounds were rescored to confirm the accuracy of binding using another scoring function (x-score). The final conclusions were drawn based on the results obtained from the GOLD and x-score. Further experiments were conducted to identify the atomic level interactions of selected compounds with GCGR. PMID:26236379

  9. Automatic gene collection system for genome-scale overview of G-protein coupled receptors in eukaryotes.

    PubMed

    Ono, Yukiteru; Fujibuchi, Wataru; Suwa, Makiko

    2005-12-30

    We have developed an automatic system for identifying GPCR (G-protein coupled receptor) genes from various kinds of genomes, which is finally deposited in the SEVENS database (http://sevens.cbrc.jp/), by integrating such software as a gene finder, a sequence alignment tool, a motif and domain assignment tool, and a transmembrane helix predictor. SEVENS enables us to perform a genome-scale overview of the "GPCR universe" using sequences that are identified with high accuracy (99.4% sensitivity and 96.6% specificity). Using this system, we surveyed the complete genomes of 7 eukaryotes and 224 prokaryotes, and found that there are 4 to 1016 GPCR genes in the 7 eukaryotes, and only a total of 16 GPCR genes in all the prokaryotes. Our preliminary results indicate that 11 subfamilies of the Class A family, the Class 2(B) family, the Class 3(C) family and the fz/smo family are commonly found among human, fly, and nematode genomes. We also analyzed the chromosomal locations of the GPCR genes with the Kolmogorov-Smirnov test, and found that species-specific families, such as olfactory, taste, and chemokine receptors in human and nematode chemoreceptor in worm, tend to form clusters extensively, whereas no significant clusters were detected in fly and plant genomes.

  10. Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

    PubMed

    Pal, Kasturi; Hwang, Sun-Hee; Somatilaka, Bandarigoda; Badgandi, Hemant; Jackson, Peter K; DeFea, Kathryn; Mukhopadhyay, Saikat

    2016-03-28

    Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment. PMID:27002170

  11. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81.

    PubMed

    Liu, Changlu; Wu, Jiejun; Zhu, Jessica; Kuei, Chester; Yu, Jingxue; Shelton, Jonathan; Sutton, Steven W; Li, Xiaorong; Yun, Su Jin; Mirzadegan, Taraneh; Mazur, Curt; Kamme, Fredrik; Lovenberg, Timothy W

    2009-01-30

    Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.

  12. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells

    PubMed Central

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-01-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80] PMID:25413305

  13. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    PubMed

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes. PMID:25118328

  14. Novel computational methodologies for structural modeling of spacious ligand binding sites of G-protein-coupled receptors: development and application to human leukotriene B4 receptor.

    PubMed

    Ishino, Yoko; Harada, Takanori

    2012-01-01

    This paper describes a novel method to predict the activated structures of G-protein-coupled receptors (GPCRs) with high accuracy, while aiming for the use of the predicted 3D structures in in silico virtual screening in the future. We propose a new method for modeling GPCR thermal fluctuations, where conformation changes of the proteins are modeled by combining fluctuations on multiple time scales. The core idea of the method is that a molecular dynamics simulation is used to calculate average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the picosecond or nanosecond time scale, and then evolutionary computation including receptor-ligand docking simulations functions to determine the rotation angle of each helix of a GPCR protein as a movement on a longer time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the proposed method was able to derive the appropriate 3D structure of the active-state GPCR which docks with its agonists.

  15. Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell

    PubMed Central

    1995-01-01

    Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell

  16. Alteration in contractile G-protein coupled receptor expression by moist snus and nicotine in rat cerebral arteries

    SciTech Connect

    Sandhu, Hardip; Xu Cangbao; Edvinsson, Lars

    2011-04-15

    The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET{sub B}, serotonin 5-HT{sub 1B}, and thromboxane A{sub 2} TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24 h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25 ng nicotine/ml). A high dose (250 ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c, 5-HT{sub 1B} receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET{sub B}, 5-HT{sub 1B}, and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25 ng nicotine/ml) lowered the 5-HT{sub 1B} receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250 ng nicotine/ml) increased the ET{sub B} receptor-mediated contraction. The combined 5-HT{sub 1B} and 5-HT{sub 2A} receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or

  17. Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons.

    PubMed

    Quaynor, Samuel; Hu, Lian; Leung, Po Ki; Feng, Hao; Mores, Nadia; Krsmanovic, Lazar Z; Catt, Kevin J

    2007-12-01

    The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.

  18. Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Duc, Nguyen Minh; Du, Yang; Thorsen, Thor S.; Lee, Su Youn; Zhang, Cheng; Kato, Hideaki; Kobilka, Brian K.; Chung, Ka Young

    2015-05-01

    G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

  19. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

    PubMed

    Fridlyand, Leonid E; Philipson, Louis H

    2016-01-01

    Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger

  20. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis

    PubMed Central

    Fridlyand, Leonid E.; Philipson, Louis H.

    2016-01-01

    Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger

  1. Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma

    PubMed Central

    2013-01-01

    Background Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. Results We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. Conclusions Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These

  2. Spectral methods for study of the G-protein-coupled receptor rhodopsin: I. Vibrational and electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2015-05-01

    Here we review the application of modern spectral methods for the study of G-protein-coupled receptors (GPCRs) using rhodopsin as a prototype. Because X-ray analysis gives us immobile snapshots of protein conformations, it is imperative to apply spectroscopic methods for elucidating their function: vibrational (Raman, FTIR), electronic (UV-visible absorption, fluorescence) spectroscopies, and magnetic resonance (electron paramagnetic resonance, EPR), and nuclear magnetic resonance (NMR). In the first of the two companion articles, we discuss the application of optical spectroscopy for studying rhodopsin in a membrane environment. Information is obtained regarding the time-ordered sequence of events in rhodopsin activation. Isomerization of the chromophore and deprotonation of the retinal Schiff base leads to a structural change of the protein involving the motion of helices H5 and H6 in a pH-dependent process. Information is obtained that is unavailable from X-ray crystallography, which can be combined with spectroscopic studies to achieve a more complete understanding of GPCR function.

  3. New insights into sodium transport regulation in the distal nephron: Role of G-protein coupled receptors

    PubMed Central

    Morla, Luciana; Edwards, Aurélie; Crambert, Gilles

    2016-01-01

    The renal handling of Na+ balance is a major determinant of the blood pressure (BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part (i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na+ excretion and are the target of many different regulatory processes that modulate Na+ retention more or less efficiently. G-protein coupled receptors (GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na+ excretion, this review also highlights the complexity of these different pathways, and the connections between them. PMID:26981195

  4. Oleic acid enhances G protein coupled receptor 43 expression in bovine intramuscular adipocytes but not in subcutaneous adipocytes.

    PubMed

    Chung, K Y; Smith, S B; Choi, S H; Johnson, B J

    2016-05-01

    We hypothesized that fatty acids would differentially affect G protein coupled receptor (GPR) 43 mRNA expression and GPR43 protein concentrations in bovine intramuscular (IM) and subcutaneous (SC) adipocytes. The GPR43 protein was detected in bovine liver, pancreas, and semimembranosus (MUS) muscle in samples taken at slaughter. Similarly, GPR43 protein levels were similar in IM adipose tissue and SM muscle but was barely detectable in SC adipose tissue. Primary cultures of IM and SC stromal vascular cells were isolated from bovine adipose tissues. Oleic acid (100 μ) stimulated PPARγ gene expression and decreased stearoyl-CoA desaturase (SCD) gene expression but had no effect on GPR43 gene expression, which was readily detectable in both IM and SC adipocytes. Differentiation cocktail (Diff; 10 μ insulin, 4 μ dexamethasone, and 10 μ ciglitizone) stimulated CCAAT/enhancer-binding protein β (C/EBPβ) and PPARγ gene expression in SC but not IM adipocytes, but Diff increased SCD gene expression in both cell types. Linoleic acid (10 µ) increased PPARγ gene expression relative to Diff cocktail in SC adipocytes, whereas linoleic acid and α-linolenic decreased SCD gene expression relative to control adipocytes and adipocytes incubated with Diff ( < 0.05). Increasing concentrations of oleic acid (1, 10, 100, and 500 μM) increased GPR43 protein and mRNA expression in IM but not SC adipocytes. These data indicated that oleic acid alters mRNA and protein concentrations of GPR43 in bovine IM adipocytes. PMID:27285685

  5. Reduced Food Intake and Body Weight in Mice Deficient for the G Protein-Coupled Receptor GPR82

    PubMed Central

    Teupser, Daniel; Holdt, Lesca Miriam; Tönjes, Anke; Kern, Matthias; Dietrich, Kerstin; Kovacs, Peter; Krügel, Ute; Scheidt, Holger A.; Schiller, Jürgen; Huster, Daniel; Brockmann, Gudrun A.; Augustin, Martin; Thiery, Joachim; Blüher, Matthias; Stumvoll, Michael; Schöneberg, Torsten; Schulz, Angela

    2011-01-01

    G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments. PMID:22216272

  6. Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

    DOE PAGESBeta

    Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu; Chawla, Udeep; Mamontov, Eugene; Brown, Michael F.; Chu, Xiang -Qiang

    2016-09-15

    Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

  7. Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

    SciTech Connect

    Thal, David M.; Homan, Kristoff T.; Chen, Jun; Wu, Emily K.; Hinkle, Patricia M.; Huang, Z. Maggie; Chuprun, J. Kurt; Song, Jianliang; Gao, Erhe; Cheung, Joseph Y.; Sklar, Larry A.; Koch, Walter J.; Tesmer, John J.G.

    2012-08-10

    G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

  8. Current Techniques for Studying Oligomer Formations of G-Protein-Coupled Receptors Using Mammalian and Yeast Cells.

    PubMed

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2016-05-27

    G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques. PMID:27052183

  9. G Protein-Coupled Receptors (GPCRs) in Alzheimer’s Disease: A Focus on BACE1 Related GPCRs

    PubMed Central

    Zhao, Juan; Deng, Yulin; Jiang, Zhaotan; Qing, Hong

    2016-01-01

    The G protein coupled receptors (GPCRs) have been considered as one of the largest families of validated drug targets, which involve in almost overall physiological functions and pathological processes. Meanwhile, Alzheimer’s disease (AD), the most common type of dementia, affects thinking, learning, memory and behavior of elderly people, that has become the hotspot nowadays for its increasing risks and incurability. The above fields have been intensively studied, and the link between the two has been demonstrated, whereas the way how GPCRs perturb AD progress are yet to be further explored given their complexities. In this review, we summarized recent progress regarding the GPCRs interacted with β-site APP cleaving enzyme 1 (BACE1), a key secretase in AD pathogenesis. Then we discussed the current findings on the regulatory roles of GPCRs on BACE1, and the possibility for pharmaceutical treatment of AD patients by the allosteric modulators and biased ligands of GPCRs. We hope this review can provide new insights into the understanding of mechanistic link between GPCRs and BACE1, and highlight the potential of GPCRs as therapeutic target for AD. PMID:27047374

  10. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus

    PubMed Central

    Li, Ting; Cao, Chuanwang; Yang, Ting; Zhang, Lee; He, Lin; Xi, Zhiyong; Bian, Guowu; Liu, Nannan

    2015-01-01

    Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes’ resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes. PMID:26656663

  11. The Conserved G-Protein Coupled Receptor FSHR-1 Regulates Protective Host Responses to Infection and Oxidative Stress

    PubMed Central

    Giannini, Jennifer A.; Robinson, Joseph D.; Powell, Jennifer R.

    2015-01-01

    The innate immune system’s ability to sense an infection is critical so that it can rapidly respond if pathogenic microorganisms threaten the host, but otherwise maintain a quiescent baseline state to avoid causing damage to the host or to commensal microorganisms. One important mechanism for discriminating between pathogenic and non-pathogenic bacteria is the recognition of cellular damage caused by a pathogen during the course of infection. In Caenorhabditis elegans, the conserved G-protein coupled receptor FSHR-1 is an important constituent of the innate immune response. FSHR-1 activates the expression of antimicrobial infection response genes in infected worms and delays accumulation of the ingested pathogen Pseudomonas aeruginosa. FSHR-1 is central not only to the worm’s survival of infection by multiple pathogens, but also to the worm’s survival of xenobiotic cadmium and oxidative stresses. Infected worms produce reactive oxygen species to fight off the pathogens; FSHR-1 is required at the site of infection for the expression of detoxifying genes that protect the host from collateral damage caused by this defense response. Finally, the FSHR-1 pathway is important for the ability of worms to discriminate pathogenic from benign bacteria and subsequently initiate an aversive learning program that promotes selective pathogen avoidance. PMID:26360906

  12. Peripheral serotonin-mediated system suppresses bone development and regeneration via serotonin 6 G-protein-coupled receptor

    PubMed Central

    Yun, Hyung-Mun; Park, Kyung-Ran; Hong, Jin Tae; Kim, Eun-Cheol

    2016-01-01

    Serotonin is important in brain functions and involved in neurological diseases. It is also drawn considerable attention in bone disease since it mainly produced by the gut. Serotonin 6 G-protein-coupled receptor (5-HT6R) is clinical targets for the treatment of neurological diseases. However, 5-HT6R as a therapeutic target in bone has not been reported. Herein, we found that 5-HT6R showed higher expression in bone, and its expression was increased during bone remodeling and osteoblast differentiation. The activation of 5-HT6R by ST1936 caused the inhibition of ALP activity and mineralization in primary osteoblast cultures, which was antagonized by SB258585, an antagonist and by the knockdown of 5-HT6R. Further investigation indicated that 5-HT6R inhibited osteoblast differentiation via Jab1 in BMP2 signaling but not PKA and ERK1/2. In vivo studies showed that the activation of 5-HT6R inhibited bone regeneration in the calvarial defect mice and also delayed bone development in newborn mice; this response was antagonized by SB258585. Therefore, our findings indicate a key role of 5-HT6R in bone formation through serotonin originating in the peripheral system, and suggest that it is a novel therapeutic target for drug development in the bone repair and bone diseases. PMID:27581523

  13. Discovery and Cardioprotective Effects of the First Non-Peptide Agonists of the G Protein-Coupled Prokineticin Receptor-1

    PubMed Central

    Urayama, Kyoji; Nishi, Toshishide; Kurose, Hitoshi; Tafi, Andrea; Ribeiro, Nigel; Désaubry, Laurent; Nebigil, Canan G.

    2015-01-01

    Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI. PMID:25831128

  14. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  15. Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue.

    PubMed

    Amisten, Stefan

    2016-01-01

    G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue. PMID:26928540

  16. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    SciTech Connect

    Shi, Yanan; Liu, Xiaochun; Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y.; Cheng, Shuk Han; Li, Shuisheng; Zhang, Yong; Cheng, Christopher H.K.; Lin, Haoran

    2013-05-24

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

  17. Peripheral serotonin-mediated system suppresses bone development and regeneration via serotonin 6 G-protein-coupled receptor.

    PubMed

    Yun, Hyung-Mun; Park, Kyung-Ran; Hong, Jin Tae; Kim, Eun-Cheol

    2016-01-01

    Serotonin is important in brain functions and involved in neurological diseases. It is also drawn considerable attention in bone disease since it mainly produced by the gut. Serotonin 6 G-protein-coupled receptor (5-HT6R) is clinical targets for the treatment of neurological diseases. However, 5-HT6R as a therapeutic target in bone has not been reported. Herein, we found that 5-HT6R showed higher expression in bone, and its expression was increased during bone remodeling and osteoblast differentiation. The activation of 5-HT6R by ST1936 caused the inhibition of ALP activity and mineralization in primary osteoblast cultures, which was antagonized by SB258585, an antagonist and by the knockdown of 5-HT6R. Further investigation indicated that 5-HT6R inhibited osteoblast differentiation via Jab1 in BMP2 signaling but not PKA and ERK1/2. In vivo studies showed that the activation of 5-HT6R inhibited bone regeneration in the calvarial defect mice and also delayed bone development in newborn mice; this response was antagonized by SB258585. Therefore, our findings indicate a key role of 5-HT6R in bone formation through serotonin originating in the peripheral system, and suggest that it is a novel therapeutic target for drug development in the bone repair and bone diseases. PMID:27581523

  18. The Conserved G-Protein Coupled Receptor FSHR-1 Regulates Protective Host Responses to Infection and Oxidative Stress.

    PubMed

    Miller, Elizabeth V; Grandi, Leah N; Giannini, Jennifer A; Robinson, Joseph D; Powell, Jennifer R

    2015-01-01

    The innate immune system's ability to sense an infection is critical so that it can rapidly respond if pathogenic microorganisms threaten the host, but otherwise maintain a quiescent baseline state to avoid causing damage to the host or to commensal microorganisms. One important mechanism for discriminating between pathogenic and non-pathogenic bacteria is the recognition of cellular damage caused by a pathogen during the course of infection. In Caenorhabditis elegans, the conserved G-protein coupled receptor FSHR-1 is an important constituent of the innate immune response. FSHR-1 activates the expression of antimicrobial infection response genes in infected worms and delays accumulation of the ingested pathogen Pseudomonas aeruginosa. FSHR-1 is central not only to the worm's survival of infection by multiple pathogens, but also to the worm's survival of xenobiotic cadmium and oxidative stresses. Infected worms produce reactive oxygen species to fight off the pathogens; FSHR-1 is required at the site of infection for the expression of detoxifying genes that protect the host from collateral damage caused by this defense response. Finally, the FSHR-1 pathway is important for the ability of worms to discriminate pathogenic from benign bacteria and subsequently initiate an aversive learning program that promotes selective pathogen avoidance. PMID:26360906

  19. In vivo stoichiometry monitoring of G protein coupled receptor oligomers using spectrally resolved two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Stoneman, M. R.; Singh, D. R.; Raicu, V.

    2010-02-01

    Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the cell under study is scanned a number of times in order to accumulate enough spectral information to accurately determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor, Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.

  20. Mechanism for Adhesion G Protein-Coupled Receptor GPR56-Mediated RhoA Activation Induced By Collagen III Stimulation

    PubMed Central

    Luo, Rong; Jeong, Sung-Jin; Yang, Annie; Wen, Miaoyun; Saslowsky, David E.; Lencer, Wayne I.; Araç, Demet; Piao, Xianhua

    2014-01-01

    GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family. PMID:24949629