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At least 109 records · Page 6Linked to original sources
PubMed2004

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila.

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted-Methuselah system is involved in the control of animal ageing.

Aging↗
PubMed2006

Asymmetric conformational changes in a GPCR dimer controlled by G-proteins.

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo- or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B(4) receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

Dimerization↗
PubMed2006

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR.

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.

Amino Acid Sequence↗
PubMed2004

GPCR-interacting proteins (GIPs): nature and functions.

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Developing chemokine mutants with improved proteoglycan affinity and knocked-out GPCR activity as anti-inflammatory recombinant drugs.

The interaction of chemokines and GAGs (glycosaminoglycans) on endothelial surfaces is a crucial step for establishing a chemotactic gradient which leads to the functional presentation of chemokines to their GPCRs (G-protein-coupled receptors) and thus to activation of approaching leucocytes. Based on molecular modelling, biophysical investigations, cell-based and in vivo experiments, we have developed a novel concept for therapeutically interfering with chemokine-GAG interactions, namely dominant-negative chemokine mutants with improved GAG binding affinity and knocked-out GPCR activity. These recombinant proteins displace their wild-type chemokine counterparts from the natural proteoglycan co-receptors without being able to activate leucocytes via GPCRs. Our mutant chemokines therefore represent the first protein-based GAG antagonists with high therapeutic potential in inflammatory diseases.

Amino Acid Motifs↗
PubMed1999

Ig-hepta, a novel member of the G protein-coupled hepta-helical receptor (GPCR) family that has immunoglobulin-like repeats in a long N-terminal extracellular domain and defines a new subfamily of GPCRs.

A novel member of the G protein-coupled receptor (GPCR) family was cloned and characterized, which is unique, among the members, in its long extracellular domain comprising Ig-like repeats and in its high expression predominantly in the lung. The clone (Ig-Hepta) was first identified as a polymerase chain reaction product generated with primers designed to amplify secretin receptor family members including the parathyroid hormone-related peptide receptors. Analysis of the open reading frame of cDNAs isolated from a rat lung cDNA library indicated that Ig-Hepta is a protein of 1389 amino acid residues and has two Ig-like repeats in the N-terminal extracellular domain (exodomain) of 1053 amino acid residues and 7 transmembrane spans in the C-terminal region. Northern blot analysis revealed very high expression of its mRNA in the lung and low but detectable levels in the kidney and heart. The mRNA expression in the lung was found to be strongly induced postnatally. Biochemical analysis indicated that Ig-Hepta is a highly glycosylated protein and exists as a disulfide-linked dimer. Immunohistochemistry on rat lung and kidney sections revealed dense localization of Ig-Hepta in alveolar walls and intercalated cells in the collecting duct, respectively, suggesting a role in the regulation of acid-base balance. Ig-Hepta defines a new subfamily of GPCRs.

Amino Acid Sequence↗
PubMed2005

Teaching resources. Introduction: Overview of pathways and networks and GPCR signaling.

This Teaching Resource provides the overview to the course "Cell Signaling Systems: A Course for Graduate Students" and lays out the general principles that can be deduced from the current understanding of the organization of cell signaling pathways and networks and how information flows through these pathways and networks. In addition, the lecture provides an overview of G protein-coupled receptor (GPCR) signaling. A description of the lecture, along with a set of slides (http://stke.sciencemag.org/cgi/content/full/sigtrans;2005/270/tr4/DC1) used to present this information, is provided.

Animals↗
PubMed2005

Distribution of G-protein-coupled receptor (GPCR)135 binding sites and receptor mRNA in the rat brain suggests a role for relaxin-3 in neuroendocrine and sensory processing.

G-protein-coupled receptor 135 (GPCR135), a former orphan GPCR also known as SALPR, has recently been shown to be modulated by relaxin-3 (R3). In addition to GPCR135, R3 has been shown to be an agonist for GPCR142 (which is a pseudogene in the rat) and to activate LGR7, which is primarily the receptor for relaxin-1/2. The interaction of R3 with LGR7 has confounded the autoradiographic study of the GPCR135 distribution in the rat CNS due to significant expression of LGR7 in the brain. R3/I5, a chimera of the B-chain of R3 bonded to the A-chain of INSL-5, is a specific GPCR135 agonist which is highly selective for GPCR135 over LGR7. [(125)I]R3/I5 specifically binds to sites on rat brain sections with a pharmacology matching results from membrane preparations of recombinant GPCR135 receptors. Autoradiographic studies show the GPCR135 receptor density is most prominent in areas such as the olfactory bulb, sensory cortex, amygdala, thalamus, paraventricular nucleus, supraoptic nucleus, inferior and superior colliculus. The GPCR135 mRNA distribution generally overlaps the pattern of GPCR135 binding sites shown by autoradiography using [(125)I]R3/I5. The nucleus incertus, which has been implicated in the extrapituitary actions of corticotropin-releasing hormone, is the primary source of R3 in the rat central nervous system and expresses GPCR135 receptors. These binding autoradiography and in situ hybridization data suggest that GPCR135 plays an important role in the central processing of sensory signals in rats, are consistent with a putative role for R3/GPCR135 as modulators of stress responses, and confirm the identity of R3 as the central nervous system ligand for GPCR135.

Animals↗
PubMed2006

G-protein-coupled receptor (GPCR)-142 does not contribute to relaxin-3 binding in the mouse brain: further support that relaxin-3 is the physiological ligand for GPCR135.

Relaxin-3 is a recently discovered member of the insulin/relaxin superfamily that has been shown to be the endogenous ligand for G-protein-coupled receptor (GPCR)135 (SALPR). In addition, relaxin-3 has demonstrated affinity and functional agonism for GPCR142 (GPR100) and LGR7 receptors in vitro. Recent evidence suggests GPCR142 is the insulin-like peptide 5 (INSL5) receptor and LGR7 is the actual relaxin receptor. We have recently described a chimeric R3/I5 peptide that selectively activates GPCR135 and GPCR142, but lacks affinity for LGR7. GPCR142 is a pseudogene in the rat, which allowed the use of [(125)I]-R3/I5 to show GPCR135-like binding sites in the rat central nervous system by autoradiography. However, mouse GPCR142 is a viable gene. In the present study we explore whether GPCR142 is expressed in the mouse brain and whether it is likely to contribute to or interfere with the pharmacological evaluation of relaxin-3 ligands. Competition binding studies confirmed mINSL5 and [(125)I]-mINSL5 bind to mGPCR142 with high affinity. However, no detectable specific [(125)I]-mINSL5 binding sites were detected throughout the mouse brain and unlabelled INSL5 did not displace [(125)I]-R3/I5 binding sites, indicating an absence of detectable GPCR142 binding sites. Consistent with these findings, neither GPCR142 nor INSL5 mRNA were detectable in mouse brain by in situ hybridization. Overall, the distribution of GPCR135 mRNA overlapped with the distribution of GPCR135 binding sites shown by autoradiography using [(125)I]-R3/I5. GPCR135 mRNA and GPCR135 receptor binding sites are most prominent in the mouse amygdala and hypothalamus. These data suggest that relaxin-3/GPCR135 is the receptor ligand pair with physiological relevance in mouse brain.

Amino Acid Sequence↗
PubMed2004

Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.

G2A is a G-protein-coupled receptor (GPCR) involved in immune regulation. Previous studies have shown that lysophosphatidylcholine (LPC), a bioactive lipid associated with atherosclerosis and autoimmunity, acts through G2A to induce diverse biologic effects. Production of LPC during cell apoptosis serves as a chemotactic signal for macrophage recruitment. Here we demonstrate that macrophage chemotaxis to LPC is dependent on G2A function. Wild-type but not G2A-deficient mouse peritoneal macrophages migrated toward LPC. RNAi-mediated knockdown of G2A in J774A.1 macrophages abolished LPC-induced chemotaxis, whereas overexpression of G2A significantly enhanced this process. Mutation of the conserved DRY motif of G2A resulted in loss of chemotaxis to LPC, suggesting a requirement for G-protein signaling. Unlike most GPCRs, including the chemokine receptors, coupling to G(i) is not required for LPC/G2A-mediated chemotaxis, but coupling to G(q/11) and G(12/13) is necessary as judged by inhibition with dominant negative forms of these alpha subunits or with regulators of G-protein signaling (RGS) constructs. Collectively, these data establish that pertussis toxin-insensitive G2A signaling regulates macrophage chemotaxis to LPC. Defects in this signaling pathway may be related to the pathogenesis of systemic autoimmune disease.

Animals↗
PubMed2005

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

Animals↗
PubMed2006

Assessment of degradation and ubiquitination of CXCR4, a GPCR regulated by EGFR family members.

The ErbB2/HER2 receptors are aberrantly expressed in certain mammary epithelial cancers. A recent study has shown that in a subset of these breast cancers, the HER2 receptors contribute to increased cell surface levels of the chemokine receptor CXCR4, which in turn results in increased metastasis of the breast cancers. Therefore, information concerning the mechanisms regulating CXCR4 receptors levels is essential to our understanding of its role in cancer cell metastasis. CXCR4 is a member of the G protein-coupled receptor (GPCR) family, and herein we describe methods to monitor the ubiquitination, degradation, and downregulation of CXCR4.

ErbB Receptors↗
PubMed2006

Ligand-based homology modeling as attractive tool to inspect GPCR structural plasticity.

G protein-coupled receptors (GPCRs) represent the largest family known of signal-transducing molecules. They convey signals for light and many extracellular regulatory molecules. GPCRs have been found to be dysfunctional/dysregulated in a growing number of human diseases and they have been estimated to be the targets of more than 40% of the drugs used in clinical medicine today. The crystal structure of rhodopsin provides the first three-dimensional GPCR information, which now supports homology modeling studies and structure-based drug design approaches. Here, we review our recent work on adenosine receptors, a family of GPCRs and, in particular, on A(3) adenosine receptor subtype antagonists. We will focus on an alternative approach to computationally explore the multi-conformational space of the antagonist-like state of the human A(3) receptor. We define ligand-based homology modeling as new approach to simulate the reorganization of the receptor induced by the ligand binding. The success of this approach is due to the synergic interaction between theory and experiment.

Adenosine A3 Receptor Antagonists↗
PubMed2004

The selection and design of GPCR ligands: from concept to the clinic.

Virtual screening methods using structure-based, pharmacophore-based and descriptor based protocols may be used to identify ligands for the G-protein coupled receptor target family. A complementary approach is the synthesis and screening of compound libraries designed using privileged motifs and/or based on validated hit molecules. A virtual screening approach based on molecular docking performed with GOLD using a templated homology model and a consensus scoring procedure can identify vasopressin 1a receptor antagonists. In a separate project a library design and synthesis approach based around validated hit GPCR ligands led to the identification of potent oxytocin antagonists. Subsequent optimisation of the initial library compounds has provided compounds that are now being evaluated in the clinic for the treatment of preterm labour.

Animals↗
PubMed2004

The different strategies for designing GPCR and kinase targeted libraries.

In recent years the trend in combinatorial library design has shifted to include target class focusing along with diversity and drug-likeness criteria. In this manuscript we review the computational tools available for target class library design and highlight the areas where they have proven useful in our work. The protein kinase family is used to illustrated structure-based target class focused library design, and the G-protein coupled receptor (GPCR) family is used to illustrate ligand-based target class focused library design. Most of the tools discussed are those designed for libraries targeted to a single protein and are simply applied "brute-force" to a large number of targets within the family. The tools that have proven to be the most useful in our work are those that can extract trends from the computational data such as docking and clustering or data mining large amounts of structure activity or high throughput screening data. Finally, areas where improvements are needed in the computational tools available for target class focusing are highlighted. These areas include tools to extract the relevant patterns from all available information for a family of targets, tools to efficiently apply models for all targets in the family rather than just a small subset, mining tools to extract the relevant information from the computational absorption, distribution, metabolism, excretion and toxicity (ADMET) and targeting data, and tools to allow interactive exploration of the virtual space around a library to facilitate the selection of the library that best suits the needs of the design team.

Animals↗
PubMed2004

Orphan GPCR target validation.

Nearly half of the identified non-olfactory G protein-coupled receptors (GPCRs) have no identified cognate ligand. These 'orphan' receptors are likely to have important physiological roles that could potentially be exploited therapeutically. However, by definition, such receptors are not immediately open for pharmacological investigation of their function. Here we summarize several strategic approaches to facilitate the discovery of orphan GPCR biology.

Animals↗