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At least 91 records · Page 5Linked to original sources
PubMed2006

DEP-domain-mediated regulation of GPCR signaling responses.

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response.

Amino Acid Sequence↗
PubMed2005

Pepducins: an effective means to inhibit GPCR signaling by neutrophils.

G-protein-coupled receptors (GPCRs) have a central role not only in the competent development of an innate myeloid response to foreign pathogens but, if dysregulated, might contribute to phagocyte-mediated organ injury. Here, recent findings from a study in which neutrophil trafficking is inhibited by using a novel family of GPCR signaling inhibitors, known as pepducins, are discussed.

Animals↗
PubMed2003

GPCR dimerisation.

The concept that GPCRs exist and potentially function as dimers and/or higher oligomers has progressed recently from hypothesis to being widely accepted. A range of techniques has contributed to this understanding, including co-immunoprecipitation and various forms of fluorescence and bioluminescence resonance energy transfer. Although co-immunoprecipitation studies indicate the capacity of a wide range of GPCRs to form hetero-dimers as well as homo-dimers, this approach is not well suited to examine selectivity of interactions. Both bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) have been applied to the detection of GPCR dimerisation in intact cells and BRET and FRET have been used to attempt to quantitate the fraction of GPCRs present as dimers. Following heterologous expression, a considerable fraction of many GPCRs is not fully processed and is trafficked to the proteasome or lysosome for destruction. A distinct limitation of both BRET and conventional FRET approaches is that both the energy donor and energy acceptor tags are inside the cell. Time-resolved FRET employing N-terminally epitope-tagged GPCRs has been used to allow detection only of dimers trafficked successfully to the cell surface. Reports indicating the appearance of distinct pharmacology and function following co-expression of two GPCRs are fascinating. Much remains to be examined, however, on the specificity and mechanisms of these interactions and to develop techniques to monitor the function only of hetero-dimers when the corresponding homo-dimers must also be present.

Animals↗
PubMed2005

Lipids, lipid rafts and caveolae: their importance for GPCR signaling and their centrality to the endocannabinoid system.

Scientific views of cell membrane organization are presently changing. Rather than serving only as the medium through which membrane proteins diffuse, lipid bilayers have now been shown to form compartmentalized domains with different biophysical properties (rafts/caveolae). For membrane proteins such as the G protein coupled receptors (GPCRs), a raft domain provides a platform for the assembly of signaling complexes and prevents cross-talk between pathways. Lipid composition also has a strong influence on the conformational activity of GPCRs. For certain GPCRs, such as the cannabinoid receptors, the lipid bilayer has additional significance. Endocannabinoids such as anandamide (AEA) are created in a lipid bilayer from lipid and act at the membrane embedded CB1 receptor. Endocannabinoids exiting the CB1 receptor are transported either by a carrier-mediated or a simple diffusion process to the membrane of the postsynaptic cell. Following cellular uptake, perhaps via caveolae/lipid raft-related endocytosis, AEA is rapidly metabolized by a membrane-associated enzyme, fatty acid amide hydrolase (FAAH) located in the endoplasmic reticulum. The entry point for AEA into FAAH appears to be from the lipid bilayer. This review explores the importance of lipid composition and lipid rafts to GPCR signaling and then focuses on the intimate relationship that exists between the lipid environment and the endocannabinoid system.

Amidohydrolases↗
PubMed2005

FMRFamide related peptide ligands activate the Caenorhabditis elegans orphan GPCR Y59H11AL.1.

G-protein coupled receptors (GPCRs) are ancient molecules that can sense environmental and physiological signals. Currently, the majority of the predicted Caenorhabditis elegans GPCRs are orphan. Here, we describe the characterization of such an orphan C. elegans GPCR, which is categorized in the tachykinin-like group of receptors. Since the C. elegans genome predicts only one tachykinin-like peptide (SFDRMGGTEFGLM), which could not activate the receptor, we hypothesized that one or some of the numerous FMRFamide related peptides (FaRPs) could be the cognate ligands for this receptor. This hypothesis was based on the suggestion that RFamides may be ancestral neuropeptides, from which a lot of the amidated neuropeptides, including tachykinins, derived. Indeed, we found that the orphan receptor encoded by the Y59H11AL.1 gene is activated by several C. elegans neuropeptides, including SPMERSAMVRFamide. These peptides activate the receptor in a concentration-dependent way.

Amino Acid Sequence↗
PubMed2005

Functional analysis of heterologous GPCR signalling pathways in yeast.

G protein-coupled receptors (GPCRs) regulate diverse biological processes in eukaryotes and such conservation allows an almost unrestricted interchange of signalling components between different cell types. Yeasts are attractive hosts in which to study GPCRs--they are amenable to both genetic and biochemical manipulation and their robustness, low cost and our ability to create strains that lack endogenous GPCRs make them ideal starting points for the development of assays suitable for high-throughput screening. Here we introduce readers to the possibilities of using yeast to analyse GPCRs describing the endogenous signalling pathways, the development of assays for heterologous GPCRs and the technology to elucidate GPCR structure and activity, focusing on the budding yeast Saccharomyces cerevisiae and recent developments using the fission yeast Schizosaccharomyces pombe.

Genes, Reporter↗
PubMed2004

Techniques: GPCR assembly, pharmacology and screening by flow cytometry.

Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed ('suspension array') analysis of cell responses and protein-protein interactions. Innovative sample-handling systems also provide sub-second resolution of interaction kinetics and 1 second per well throughput of microliter-sized samples from multiwell plates. Flow cytometric methods using microspheres for analysis of GPCRs that interact with intracellular and extracellular binding partners such as ligands, G proteins and kinases have been established. These analyses can produce quantitative pharmacological data analogous to radioligand assays, and, in some cases, the probes can be integrated into the assembly as fluorescent fusion proteins.

Animals↗
PubMed2005

GPCR deorphanizations: the novel, the known and the unexpected transmitters.

Pairing orphan G-protein-coupled receptors (GPCRs) to potential ligands found in tissue extracts or in libraries of synthetic compounds has identified an ever-increasing number of transmitters, some of which are novel, some of which were previously known and, more recently, some of which are unexpected. These discoveries raise questions about the nature of transmitters and the specificity of GPCRs. In this article, several aspects of the impact of orphan GPCR research on our understanding of the diversity of transmitters will be discussed.

Humans↗
PubMed2025

Decoding missense variants pleiotropy in the immune GPCR P2RY8.

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors and remain a central focus in pharmacology and drug discovery. Despite extensive structural and pharmacological studies, the functional impact of missense variation across GPCRs remains poorly understood, particularly for receptors involved in immune regulation. In this issue of Cell Genomics, LaFlam et al.1 systematically map P2RY8 variant functions using deep mutational scanning (DMS) combined with structural biology approaches, revealing pleiotropy and mechanisms linking GPCR variation to B cell confinement and lymphoma.

Humans↗
PubMed2003

Depicting a protein's two faces: GPCR classification by phylogenetic tree-based HMMs.

Related proteins with similar biological functions generally share common features, allowing us to extract the common sequence features. These common features enable us to build statistical models that can be used to classify proteins, to predict new members, and to study the sequence-function relationship of this protein function group. Although evolution underlies the basis of multiple sequence analysis methods, most methods ignore phylogenetic relationships and the evolutionary process in building these statistical models. Previously we have shown that a phylogenetic tree-based profile hidden Markov model (T-HMM) is superior in generating a profile for a group of similar proteins. In this study we used the method to generate common features of G protein-coupled receptors (GPCRs). The profile generated by T-HMM gives high accuracy in GPCR function classification, both by ligand and by coupled G protein.

Animals↗
PubMed2003

Acute oral dexamethasone administration reduces levels of orphan GPCR glucocorticoid-induced receptor (GIR) mRNA in rodent brain: potential role in HPA-axis function.

Glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor (GPCR) with predominant expression in brain and thymus. More specifically, high levels of GIR expression have been described in brain regions of mouse, rat and human including limbic forebrain and hypothalamic regions, suggesting a role for GIR in memory, cognition, stress, reward or the control of emotion. Previous in vitro studies performed in murine thymocytes demonstrated an induction of GIR following dexamethasone treatment, suggesting the potential in vivo regulation of GIR by glucocorticoids. Glucocorticoids have been implicated in a number of disorders. In this study we employed in situ hybridisation with semi-quantitative image analysis to assess the level of GIR expression in mouse brain following acute dexamethasone administration. GIR was highly expressed in the nucleus accumbens, striatum, olfactory tubercle and nuclei of the hypothalamus. Three hours following acute dexamethasone treatment (0.05 mg/kg, p.o.), levels of GIR mRNA were found to be significantly decreased in striatum (25%, P<0.05), nucleus accumbens (19%, P<0.05), olfactory tubercle (19%, P<0.05) and CA2 sub-region of the hippocampus (30%, P<0.05) compared with vehicle. Significant decreases in GIR expression were also observed in hypothalamic nuclei including the dorsomedial (48%, P<0.05) and ventrolateral (58%, P<0.05) part of the ventromedial hypothalamic nuclei, dorsomedial hypothalamic nuclei (39%, P<0.01) and arcuate nucleus (54%, P<0.05), compared with vehicle. These data demonstrate the in vivo regulation of GIR in response to glucocorticoids and suggest a potential role for GIR in mediating the response to altered levels of glucocorticoids in disease states.

Administration, Oral↗
PubMed2001

New hypotheses for the GPCR 3D arrangement based on a molecular model of the human sweet-taste receptor.

A molecular model of the human sweet-taste receptor has been inferred from superpositions of 3D maps of sweetener interaction sites (themselves previously deduced from extensive structure-activity relationship studies on highly potent sweeteners) onto three well-known G protein-coupled receptors (GPCRs)-rhodopsin, beta(2)- and alpha(2A)-adrenergic receptors-assumed to be linked by common evolutionary origins. The model gives new answers to old questions on the GPCR 3D structure, such as on the orientation and arrangement of the binding helices, their interaxial distances, radial orientations and relative heights. The model should be useful as a new approach to the rational design of drugs.

Amino Acid Sequence↗
PubMedDate not supplied

GPCR-tailored pharmacophore pattern recognition of small molecular ligands.

The goal of our work was to differentiate between patterns, which are responsible for the activity of small molecular ligands binding to G-protein coupled receptors (GPCRs) and molecules, which are pharmacologically active on other target classes. Second the aim was to go one step further and analyze the chemical space occupied by GPCR active ligands itself, to distinguish between the actives of different subclasses or even cluster ligands for single receptors. To achieve these objectives, we have built a database of small, organic molecules, which bind to GPCRs. Once this crucial foundation for pattern recognition has been laid, we needed to find a descriptor, which is able to detect the compulsory features responsible for activity within a molecule. In this matter we found that the well accepted pharmacophore descriptor served us well. Finally we needed to find a method to display the clustering or separation of the specific ligands. We found that self-organizing maps (SOMs) perform excellently in this task. We herein present the analysis of the chemical space of active compounds, depending on their biological target, the GPCRs. We will also discuss the techniques used to create the chemical spaces. The findings can be applied and have an impact at various stages of the drug discovery process.

Ligands↗
PubMedDate not supplied

An analysis of the conserved residues between halobacterial retinal proteins and G-protein coupled receptors: implications for GPCR modeling.

An alignment of the transmembrane domains of halobacterial retinal proteins (including bacteriorhodopsin) and G-protein coupled receptors (GPCRs) is presented based on the commonality of conserved residues between families. Due to the limited sequence homology displayed by these proteins, an alternative strategy is proposed for sequence alignment that correlates residues within secondary structure elements. The nonsequential alignment developed identifies three proline and two aspartates residues that share common positions and, in the former case, similar functions in the transmembrane domain. The alignment is further applied to model the packing of transmembrane helices 5 and 6 of the beta-adrenergic receptor based on the backbone coordinates of bacteriorhodopsin helices 3 and 2, respectively. Unlike models derived from standard sequential alignments, the approach developed here allows the key structural features conferred by the proline residues to be captured during model building. The structure described is also compared with available site directed mutagenesis results as well as existing GPCR models. In addition to the implications to model building, the commonality observed suggests a potential relationship among the GPCRs and retinal proteins.

Amino Acid Sequence↗
PubMed2004

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy.

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.

Cell Line↗
PubMed2005

Functional GPCR microarrays.

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.

Drug Evaluation, Preclinical↗
PubMed2006

High-resolution NMR spectroscopy of a GPCR in aligned bicelles.

Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.

Binding Sites↗
PubMed2005

Structure-based drug discovery using GPCR homology modeling: successful virtual screening for antagonists of the alpha1A adrenergic receptor.

In this paper, we describe homology modeling of the alpha1A receptor based on the X-ray structure of bovine rhodopsin. The protein model has been generated by applying ligand-supported homology modeling, using mutational and ligand SAR data to guide the protein modeling procedure. We performed a virtual screening of the company's compound collection to test how well this model is suited to identify alpha1A antagonists. We applied a hierarchical virtual screening procedure guided by 2D filters and three-dimensional pharmacophore models. The ca. 23,000 filtered compounds were docked into the alpha1A homology model with GOLD and scored with PMF. From the top-ranked compounds, 80 diverse compounds were tested in a radioligand displacement assay. 37 compounds revealed K(i) values better than 10 microM; the most active compound binds with 1.4 nM to the alpha1A receptor. Our findings suggest that rhodopsin-based homology models may be used as the structural basis for GPCR lead finding and compound optimization.

Adrenergic alpha-1 Receptor Antagonists↗