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At least 55 records · Page 3Linked to original sources
PubMed2005

Two ligands for a GPCR, proton vs lysolipid.

Recently, two different chemicals have been matched as ligands with the same G-protein-coupled receptor (GPCR). Double-pairing of OGR1 family GPCRs with proton and lysolipid raises several questions. First, whether both are the real ligands for the GPCRs. Second, whether modulation of a GPCR by two chemicals could be possible. Third, one of the chemicals is proton. Proton-sensing not only is a new action mode of GPCR activation, but also it could be generalized in other GPCRs. In this review, I would like to summarize the issue and discuss questions with pharmacological criteria.

Amino Acid Sequence↗
PubMed2005

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways.

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.

ADAM Proteins↗
PubMed2002

The use of biosensors to study GPCR function: applications for high-content screening.

Plasma membrane-associated G protein-coupled receptors (GPCRs) initiate the transmission of multiple intracellular signals leading to a myriad of physiological and pathophysiological effects. The downstream signaling events associated with occupation of the GPCR and activation of the G-protein include the generation of numerous second messenger molecules to provide the necessary signal amplification within the appropriate intracellular compartment to transmit a specific signal from the cell surface to the cell interior. The complex process of signal transmission also requires a series of highly orchestrated events which includes the translocation of cellular proteins to discreet intracellular destinations. A better understanding of these events has made it possible to design assays to examine multiple endpoints within whole cells. In this review we describe recent advances in assay biology and instrumentation useful for broadening our understanding of the molecular events associated with GPCR activation. This review will focus on novel cell-based approaches using fluorescent biosensors, such as fluorescent dyes and fluorescent protein tags, to generate information-rich data from multiple cellular targets--a process that has been referred to as high-content screening. High-content screening applications will be discussed as they pertain to specific signal transduction cascades initiated upon GPCR activation and examples of specific biosensors will be provided.

Animals↗
PubMed2005

Focused library design in GPCR projects on the example of 5-HT(2c) agonists: comparison of structure-based virtual screening with ligand-based search methods.

The aim of this study was to investigate the usefulness of structure-based virtual screening (VS) for focused library design in G protein-coupled receptors (GPCR) projects on the example of 5-HT(2c) agonists. We compared the performance of structure-based VS against two different homology models using FRED for docking and ScreenScore, FlexX, and PMF for rescoring with the results of 12 ligand-based similarity searches using four different query compounds and three different similarity metrics (Daylight, FTree, Phacir). The result of the similarity search showed much variation, from an enrichment factor up to 3.2 to worse than random, whereas the structure-based VS gave a more stable result with a constant enrichment factor around 2. Additionally, actives retrieved by the structure-based approach were more diverse than the actives among the top scorers of the similarity searches. Based on these results, we suggest basing a focused library design for a GPCR project on a combination of a ligand-based similarity search and structure-based docking.

Cluster Analysis↗
PubMed2006

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR.

Biotechnology using molecular biology, biochemistry, biophysics, and computational approaches provides an alternative approach for classical pharmacological screening to look at ligand-receptor interactions and receptor specificity, which should support the design of selective drugs based on detailed structural principles. This review addresses specific approaches to study function, structure and relevance of a major pharmaceutical target, namely the G-Protein Coupled Receptors (GPCRs). The main aim of this review has been to exploit and combine GPCR over-expression in a baculoviral expression system with solid-state MAS NMR (ssNMR) approaches for the elucidation of electronic structures of the coordinating ligands/drugs and their modes of interactions with the GPCRs. This review summarizes the approaches, possible future experiments and developments using the above combination of tools for GPCR drug discovery.

Baculoviridae↗
PubMed2005

Multi-space classification for predicting GPCR-ligands.

A classification of molecules depends on the descriptor set which is used to represent the compounds, and each descriptor could be regarded as one perception of a molecule. In this study we show that a combination of several classifiers that are grounded on separate descriptor sets can be superior to a single classifier that was built using all available descriptors. The task of predicting ligands of G-protein coupled receptors (GPCR) served as an example application. The perceptron, multilayer neural networks, and radial basis function (RBF) networks were employed for prediction. We developed classifiers with and without descriptor selection. Prediction accuracy was assessed by the area under the receiver operating characteristic (ROC) curve. In the case with descriptor selection both the selection and the rank order of the descriptors depended on the type and topology of the neural networks. We demonstrate that the overall prediction accuracy of the system can be improved by joining neural network classifiers of different type and topology using a "jury network" that is trained to evaluate the predictions from the individual classifiers. Seventy-one percent correct prediction of GPCR ligands was obtained.

Ligands↗
PubMed2006

The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling.

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.

Animals↗
PubMed2005

GPCR signaling is required for blood-brain barrier formation in drosophila.

The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. Here, we show that the G protein-coupled receptor (GPCR) Moody, the G protein subunits G alpha i and G alpha o, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. Our data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. Our study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core.

Actins↗
PubMed2005

GPCR modulation by RAMPs.

Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activity-modifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into the impact that their discovery has had on those who study GPCRs.

Animals↗
PubMed2004

GPCR expression in the heart; "new" receptors in myocytes and fibroblasts.

G protein-coupled receptor (GPCR) signaling cascades play a key role in regulation of the cardiovascular system. GPCRs comprise the largest cell-surface receptor superfamily and are also the largest class of drug targets to which agonists and antagonists are currently directed. This article reviews recent data regarding aspects of GPCR signaling and emphasizes the recent "discovery" of expression of "new" GPCRs in cardiac myocytes and fibroblasts. In addition to several well-known ("classical") GPCRs that are expressed by cardiac myocytes and fibroblasts, these cell types or cardiac tissue (cell types as-yet undefined) express a large number of other GPCRs, including receptors for peptides, lipids, and other molecules, as well as "orphan" receptors for which the natural agonists and (patho)physiologic roles in the cardiovascular system have not yet been defined. Studies of the newly recognized GPCRs-in particular, deorphanization of novel GPCRs and experiments that define their signal pathways, expression patterns, desensitization mechanisms, cross-talk with other receptors, and genetic variation-are likely to yield important new insights into cardiovascular cell function. Moreover, these "new" GPCRs are attractive targets for the development of drugs for the treatment of cardiac and other cardiovascular disorders.

Animals↗
PubMed2001

Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells.

In contrast to other families of cell surface receptors, like tyrosine kinase receptors, for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or hetero- oligomers could have important implications for the development and screening of new drugs. The major evidences supporting the idea of GPCR oligomerization come from indirect biochemical or pharmacological experiments. Here we report, using traditional co-immunoprecipitation methods, the existence of differentially epitope-tagged beta2-adrenergic receptor (beta2AR) oligomers in mammalian HEK-293 cells. Moreover, we validate the existence of receptor oligomers in living cells by a new Bioluminescence Resonance Energy Transfer (BRET) technique. Our results clearly demonstrate the presence of constitutive beta2AR oligomers in living cells that can be modulated by the selective adrenergic agonist isoproterenol, suggesting a pertinent physiological role for GPCR oligomerization.

Adrenergic beta-2 Receptor Agonists↗
PubMed2001

A compendium of specific motifs for diagnosing GPCR subtypes.

Analysis of G-protein-coupled receptor (GPCR) subtypes has attracted considerable interest because some drugs that act on GPCRs cause therapeutic problems as a result of their failure to differentiate between subtypes. In this article, an extensive compendium of diagnostic 'fingerprints' for GPCR subtypes and their families will be described. These fingerprints offer new opportunities to investigate correlations between specific sequence motifs and ligand binding or G-protein coupling, and are likely to prove valuable both in seeking novel receptors in genome data and in the characterization of orphan receptors.

Computational Biology↗
PubMed2026

Freezing with Light: Photo-Cross-Linking-Assisted Platform Enables GPCR Deorphanization.

Despite their success as drug targets, nearly one hundred G protein-coupled receptors (GPCRs) remain orphan without identified endogenous ligands. Defining these ligand-receptor pairs constitute a fundamental prerequisite for understanding receptor biology and rational drug discovery. However, deorphanization remains inherently challenging due to the transient and interface-specific ligand-GPCR interactions, especially for endogenous ones that are embedded within chemically complex environments. This In Focus article highlights a modularly designed platform that integrates site-specific photo-cross-linking reaction with proteomics to enable ligand-GPCR pairing directly in native biological contexts. Using this strategy, neuropeptide L-LEN was identified as the endogenous ligand for GPR50, forming a regulatory axis that controls energy expenditure and thermogenesis through brain-peripheral interactions.

Receptors, G-Protein-Coupled↗
PubMed2005

Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR.

G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G-protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.

Cell Line↗
PubMedDate not supplied

GPCR-induced dissociation of G-protein subunits in early stage signal transduction.

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.

Animals↗
PubMedDate not supplied

Potentiation of GPCR-signaling via membrane targeting of G protein alpha subunits.

Different assay technologies are available that allow ligand occupancy of G protein coupled receptors to be converted into robust functional assay signals. Of particular interest are universal screening systems such that activation of any GPCR can be detected with a common assay end point. The promiscuous G protein Galpha16 and chimeric G proteins are broadly used tools for setting up almost universal assay systems. Many efforts focused on making G proteins more promiscuous, however no attempts have been made to make promiscuos G proteins more sensitive by interfering with their cellular protein distribution. As a model system, we used a promiscuous G protein alphaq subunit, that lacks the highly conserved six amino acid N-terminal extension and bears four residues of alphai sequence at its C-terminus replacing the corresponding alphaq sequence (referred to as delta6qi4). When expressed in COS7 cells, delta6qi4 undergoes palmitoylation at its N-terminus. Cell fractionation and immunoblotting analysis indicated localization in the particulate and cytosolic fraction. Interestingly, introduction of a consensus site for N-terminal myristoylation (the resulting mutant referred to as delta6qi4myr) created a protein that was dually acylated and exclusively located in the particulate fraction. As a measure of G protein activation delta6qi4 and delta6qi4myr were coexpressed (in CHO cells) with a series of different Gi/o coupled receptors and ligand induced increases in intracellular Ca2+ release were determined with the FLIPR technology (Fluorescence plate imaging reader from Molecular Devices Corp.). All of the receptors interacted more efficiently with delta6qi4myr as compared with delta6qi4. It could be shown that increased functional responses of agonist activated GPCRs are due to the higher content of delta6qi4myr in the plasma membrane. Our results indicate that manipulation of subcellular localization of G protein alpha subunits-moving them from the cytosol to the plasma membrane-potentiates signaling of agonist activated GPCRs. It is concluded that addition of myristoylation sites into otherwise exclusively palmitoylated G proteins is a new and sensitive approach and may be applicable when functional assays are expected to yield weak signals as is the case when screening extracts of tissues for biologically active GPCR ligands.

Acylation↗
PubMed2003

GPCR-GRAPA-LIB--a refined library of hidden Markov Models for annotating GPCRs.

GPCR-GRAPA-LIB is a library of HMMs describing G protein coupled receptor families. These families are initially defined by class of receptor ligand, with divergent families divided into subfamilies using phylogenic analysis and knowledge of GPCR function. Protein sequences are applied to the models with the GRAPA curve-based selection criteria. RefSeq sequences for Homo sapiens, Drosophila melanogaster, and Caenorhabditis elegans have been annotated using this approach.

Algorithms↗
PubMed2004

Predicting GPCR-G-protein coupling using hidden Markov models.

MOTIVATION: Determining the coupling specificity of G-protein coupled receptors (GPCRs) is important for understanding the biology of this class of pharmacologically important proteins. Currently available in silico methods for predicting GPCR-G-protein coupling specificity have high error rate. METHOD: We introduce a new approach for creating hidden Markov models (HMMs) based on a first guess about the importance of various residues. We call these knowledge restricted HMMs to emphasize the fact that the state space of the HMM is restricted by the application of a priori knowledge. Specifically, we use only those amino acid residues of GPCRs which are likely to interact with G-proteins, namely those that are predicted to be in the intra-cellular loops. Furthermore, we concatenate these predicted loops into one sequence rather than considering them as four disparate units. This reduces the HMM state space by drastically decreasing the sequence length. RESULTS: Our knowledge restricted HMM based method to predict GPCR-G-protein coupling specificity has an error rate of <1%, when applied to a test set of GPCRs with known G-protein coupling specificity. AVAILABILITY: Academic users can get the data set mentioned herein and HMMs from the authors.

Binding Sites↗