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PubMed2005

High content screening for G protein-coupled receptors using cell-based protein translocation assays.

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

Animals↗
PubMed2006

Membrane interactive alpha-helices in GPCRs as a novel drug target.

G-Protein Coupled Receptors (GPCRs) are one of the most important targets for pharmaceutical drug design. Over the past 30 years, mounting evidence has suggested the existence of homo and hetero dimers or higher-order complexes (oligomers) that are involved in signal transduction and some diseases. The number of reports describing GPCR oligomerization has increased, and in 2003, the organization of mouse rhodopsin into two-dimensional arrays of dimers was determined by an atomic force microscopic analysis. The analysis of the mouse rhodopsin complex has enabled us to discuss the oligomerization based on structural data. Although many unsolved problems still remains, the idea that GPCRs directly interact to form oligomers has been gradually accepted. One of the recent findings in the GPCR investigations is the clarification of the mechanisms of GPCR oligomerization at a molecular level. Most of these studies have suggested the importance of transmembrane alpha-helices for GPCR oligomerization. In this review, we will first summarize the importance of GPCR oligomerization and the functions of GPCRs. Then, we will explain the involvement of transmembrane alpha-helices in the oligomerization and a drug design strategy that targets these regions for GPCR oligomerization. Considering the current drug design methods, which are based on the modification of the protein-protein interactions of soluble regions of proteins, a "peptide mimic approach" that targets the transmembrane alpha-helices constituting the interfaces would be promising in drug discovery for GPCR oligomerization. For that purpose, we must know the positions of the interfaces. However, problems specific to membrane proteins have made it difficult to identify the positions of the interfaces experimentally. Therefore, information about the interfaces predicted by bioinformatics approaches is valuable. At the end of this review, several bioinformatics approaches toward interface prediction for oligomerization are introduced. The benefits and the pitfalls of these approaches are also discussed.

Biopolymers↗
PubMed1993

The receptor revolution--multiplicity of G-protein-coupled receptors.

The superfamily of G-protein-coupled receptors (GPCR) is probably the largest protein-encoding gene family in our genome. It is already known to include hundreds of members and many more are expected to emerge as the molecular cloning revolution proceeds. By definition the GPCR respond to ligands by interacting with intracellular G-proteins and thereby transduce external signals to the interior of the cell. A large body of evidence suggests that the GPCR are organized in the cell membrane like bacteriorhodopsin (BR). All GPCR possess seven hydrophobic membrane-spanning segments which seem to form a characteristic BR-like barrel structure. Thus, the three-dimensional structure of BR may be used as a framework for computer-aided structural modelling of GPCR. The ligands which activate the various members of the GPCR family include an enormous variety of molecules such as amines, amino acids and peptides as well as several small hydrophobic molecules. Many ligands bind to multiple distinct GPCR, e.g. neuropeptide Y (NPY). We have isolated molecular clones encoding a human NPY receptor whose binding properties conform to those of the Y1 subtype. This clone will be a useful tool in our efforts to unravel the molecular mechanisms of the many physiological functions of neuropeptide Y.

Amino Acid Sequence↗
PubMed2002

Drug design strategies for targeting G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) form a large protein family that plays an important role in many physiological and pathophysiological processes. Since the sequencing of the human genome has revealed several hundred new members of this receptor family, many new opportunities for developing novel therapeutics have emerged. The increasing knowledge of GPCRs (biological target space) and their ligands (chemical ligand space) enables novel drug design strategies to accelerate the finding and optimization of GPCR leads: The crystal structure of rhodopsin provides the first three-dimensional GPCR information, which now supports homology modeling studies and structure-based drug design approaches within the GPCR target family. On the other hand, the classical ligand-based design approaches (for example, virtual screening, pharmacophore modeling, quantitative structure-activity relationship (QSAR)) are still powerful methods for lead finding and optimization. In addition, the cross-target analysis of GPCR ligands has revealed more and more common structural motifs and three-dimensional pharmacophores. Such GPCR privileged structural motifs have been successfully used by many pharmaceutical companies to design and synthesize combinatorial libraries, which are subsequently tested against novel GPCR targets for lead finding. In the near future structural biology and chemogenomics might allow the mapping of the ligand binding to the receptor. The linking of chemical and biological spaces will aid in generating lead-finding libraries, which are tailor-made for their respective receptor.

Amino Acid Motifs↗
PubMed1997

Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer.

Using the technique of differential cDNA library screening, a cDNA clone was isolated from an estrogen receptor (ER)-positive breast carcinoma cell line (MCF7) cDNA library based upon the overexpression of this gene compared to an ER-negative cell line (MDA-MB-231). Sequence analysis of this clone determined that it shared significant homology to G-protein-coupled receptors. This receptor, GPCR-Br, was abundantly expressed in the ER-positive breast carcinoma cell lines MCF7, T-47D, and MDA-MB-361. Expression was absent or minimal in the ER-negative breast carcinoma cell lines BT-20, MDA-MB-231, and HBL-100. GPCR-Br was ubiquitously expressed in human tissues examined but was most abundant in placenta. GPCR-Br expression was examined in 11 primary breast carcinomas. GPCR-Br was detected in all 4 ER-positive tumors and only 1 of 7 ER-negative tumors. Based upon PCR analysis in hybrid cell lines, the gene for GPCR-Br (HGMW-approved symbol GPR30) was mapped to chromosome 7p22. The pattern of expression of GPCR-Br indicates that this receptor may be involved in physiologic responses specific to hormonally responsive tissues.

Amino Acid Sequence↗
PubMed2001

Transactivation: a novel signaling pathway from angiotensin II to tyrosine kinase receptors.

Angiotensin II (Ang II), an octapeptide pressor hormone, activates cellular events that may contribute to the pathogenesis of cardiovascular disease. The physiological actions of Ang II are mediated via the Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R), which are G protein-coupled receptors (GPCR). GPCR share a common basic structure of seven transmembrane helices connected by alternating cytoplasmic and extracellular loops. GPCR lack intrinsic kinase activity possessed by receptor tyrosine kinases (RTK) such as platelet-derived growth factor receptor (PDGFR) or epidermal growth factor receptor (EGFR). Nonetheless, the signal transduction events activated by the AT1R mimic those of RTKs. Recently, cross-talk between GPCR and RTK has been observed. There is accumulating evidence that GPCR take advantage of signaling pathways downstream of RTK to exert its effect on the cells. In this context, RTK may be considered as one of signaling molecules downstream of GPCR.

Angiotensin II↗
PubMed1993

Sequence homology between bacteriorhodopsin and G-protein coupled receptors: exon shuffling or evolution by duplication?

Bacteriorhodopsin (BR) is a membrane protein of known structure, widely used for the homology modeling of G-protein-coupled receptors (GPCR). The observation of apparently transposed sequence similarities between some of the helical domains of BR and GPCR has led to the suggestion that exon shuffling may have occurred in the later evolution of GPCR, which would necessitate a different folding pattern for the seven transmembrane helices of GPCR. An alternate hypothesis is that duplication occurred in the evolution of an ancestral gene, such that helices 5-7 originated as duplicates of helices 1-3, leading to intragenic as well as intergenic similarities between helices 1-3 and 5-7 of BR and various GPCR. Analyses of GPCR and BR sequences suggest that such a duplication may have occurred; symmetry within the BR structure is also consistent with homology between these two regions. The hypothesis of evolution by duplication is consistent with the conventional, unshuffled homology model, which is also supported by the obvious conservation of the retinal binding Lys moiety on helix 7 in both BR and the mammalian opsins.

Amino Acid Sequence↗
PubMed2006

Regulation of G protein-coupled receptor export trafficking.

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.

Amino Acid Motifs↗
PubMed2006

G-protein coupled receptor structure.

Because of their central role in regulation of cellular function, structure/function relationships for G-protein coupled receptors (GPCR) are of vital importance, yet only recently have sufficient data been obtained to begin mapping those relationships. GPCRs regulate a wide range of cellular processes, including the senses of taste, smell, and vision, and control a myriad of intracellular signaling systems in response to external stimuli. Many diseases are linked to GPCRs. A critical need exists for structural information to inform studies on mechanism of receptor action and regulation. X-ray crystal structures of only one GPCR, in an inactive state, have been obtained to date. However considerable structural information for a variety of GPCRs has been obtained using non-crystallographic approaches. This review begins with a review of the very earliest GPCR structural information, mostly derived from rhodopsin. Because of the difficulty in crystallizing GPCRs for X-ray crystallography, the extensive published work utilizing alternative approaches to GPCR structure is reviewed, including determination of three-dimensional structure from sparse constraints. The available X-ray crystallographic analyses on bovine rhodopsin are reviewed as the only available high-resolution structures for any GPCR. Structural information available on ligand binding to several receptors is included. The limited information on excited states of receptors is also reviewed. It is concluded that while considerable basic structural information has been obtained, more data are needed to describe the molecular mechanism of activation of a GPCR.

Crystallography, X-Ray↗
PubMed2006

Regulation of alpha2AR trafficking and signaling by interacting proteins.

The continuing discovery of new G protein-coupled receptor (GPCR) interacting proteins and clarification of the functional consequences of these interactions has revealed multiple roles for these events. Some of these interactions serve to scaffold GPCRs to particular cellular micro-compartments or to tether them to defined signaling molecules, while other GPCR-protein interactions control GPCR trafficking and the kinetics of GPCR-mediated signaling transduction. This review provides a general overview of the variety of GPCR-protein interactions reported to date, and then focuses on one prototypical GPCR, the alpha(2)AR, and the in vitro and in vivo significance of its reciprocal interactions with arrestin and spinophilin. It seems appropriate to recognize the life and career of Arthur Hancock with a summary of studies that both affirm and surprise our preconceived notions of how nature is designed, as his career-long efforts similarly affirmed the complexity of human biology and attempted to surprise pathological changes in that biology with novel, discovery-based therapeutic interventions. Dr. Hancock's love of life, of family, and of commitment to making the world a better place are a model of the life well lived, and truly missed by those who were privileged to know, and thus love, him.

14-3-3 Proteins↗
PubMed2006

Targeting G protein-coupled receptor signaling in asthma.

The complex disease asthma, an obstructive lung disease in which excessive airway smooth muscle (ASM) contraction as well as increased ASM mass reduces airway lumen size and limits airflow, can be viewed as a consequence of aberrant airway G protein-coupled receptor (GPCR) function. The central role of GPCRs in determining airway resistance is underscored by the fact that almost every drug used in the treatment of asthma directly or indirectly targets either GPCR-ligand interaction, GPCR signaling, or processes that produce GPCR agonists. Although many airway cells contribute to the regulation of airway resistance and architecture, ASM properties and functions have the greatest impact on airway homeostasis. The theme of this review is that GPCR-mediated regulation of ASM tone and ASM growth is a major determinant of the acute and chronic features of asthma, and multiple strategies targeting GPCR signaling may be employed to prevent or manage these features.

Airway Resistance↗
PubMed2004

Proteome-wide classification and identification of mammalian-type GPCRs by binary topology pattern.

G protein-coupled receptors (GPCRs), a large eukaryotic protein family, have proved difficult to comprehensively detect and functionally identify by homology searches and domain detection, because they are highly divergent and their sequences share strikingly little similarity. Transmembrane (TM) topology pattern analysis has been used to classify TM proteins, and such patterns are conserved within GPCRs of similar function. Here, we developed a stepwise binary topology pattern (BTP) method for GPCR classification and identification and used it to identify and classify mammalian-type GPCRs in the genomes of 10 different eukaryotic species. A binary topology pattern was obtained for each functional class or group by assigning binary loop threshold lengths of "0" (short loop) or "1" (long loop). The GPCR-classification ability of the BTP method had quite high accuracies for classifying GPCR functions at the class level (Classes A, B, C, Frizzled/Smoothened, Non-GPCR, based on the GPCRDB classification scheme), with many classes being classified with 100% accuracy. Sufficiently high accuracies were also maintained at the functional group level, 0.945 over 15 functional groups. Proteome-wide mammalian-type GPCR searches in 10 eukaryotic genomes (H. sapiens, M. musculus, F. rubripes, C. intestinalis, A. thaliana, D. melanogaster, A. gambiae, C. elegans, P. falciparum, S. cerevisiae) using the BTP method showed much higher classification/identification in non-mammalian genomes than typical BLAST searches, in which a higher number of sequences were classified as Non-GPCR. This stepwise BTP method should prove useful for the identification and functional classification of GPCRs from the genomes of a wide range of species.

Animals↗
PubMed2004

Mutant G-protein-coupled receptors as a cause of human diseases.

G-protein-coupled receptors (GPCR) are involved in directly and indirectly controlling an extraordinary variety of physiological functions. Their key roles in cellular communication have made them the target for more than 60% of all currently prescribed drugs. Mutations in GPCR can cause acquired and inherited diseases such as retinitis pigmentosa (RP), hypo- and hyperthyroidism, nephrogenic diabetes insipidus, several fertility disorders, and even carcinomas. To date, over 600 inactivating and almost 100 activating mutations in GPCR have been identified which are responsible for more than 30 different human diseases. The number of human disorders is expected to increase given the fact that over 160 GPCR have been targeted in mice. Herein, we summarize the current knowledge relevant to understanding the molecular basis of GPCR function, with primary emphasis on the mechanisms underlying GPCR malfunction responsible for different human diseases.

Animals↗
PubMed2006

Novel phosphatidylinositol phosphate kinases with a G-protein coupled receptor signature are shared by Dictyostelium and Phytophthora.

G-protein coupled receptors (GPCR) and phosphatidylinositol phosphate kinases (PIPK) are important key switches in signal transduction pathways. A novel class of proteins was identified in the genomes of two unrelated organisms that harbor both a GPCR and a PIPK domain. Dictyostelium discoideum contains one GPCR-PIPK, which is crucial in cell-density sensing, and the genomes of Phytophthora sojae and Phytophthora ramorum each encode twelve GPCR-PIPKs. Intriguingly, these are currently the only species that have these two domains combined in one protein. Here, the structural and regulatory characteristics of GPCR-PIPKs are presented and discussed. It is hypothesized that, upon activation, GPCR-PIPKs are able to trigger heterotrimeric G-protein signaling and phosphoinositide second-messenger synthesis.

Amino Acid Sequence↗
PubMed2006

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

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.

Animals↗
PubMed1998

[Use of a G-protein-coupled receptor to communicate. An evolutionary success].

Among membrane-bound receptors, the seven transmembrane receptors are the most abundant (several thousand, 1% of the genome). They were the most successful during evolution. They are capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids, proteins, etc. They are catalysts of the GDP/GTP nucleotide exchange on heterotrimeric G proteins. They are therefore also called 'G-protein-coupled receptors' (GPCR). G proteins are composed of three subunits, G alpha and two undissociable subunits, G beta gamma. There are at least three families of GPCR showing no sequence similarity. Among G proteins, some have been crystallized (including under the heterotrimeric form) and their structure as well as their activation mechanisms are well known. The structures of GPCR are less known owing to the difficulty in crystallizing membrane-bound proteins. Indirect studies (mutations, 2D crystallization of rhodopsine, molecular modelling, etc.) lead to a useful model of the 'central core' composed of the seven transmembrane domains and of its structural modifications during activation. The intimate contact zones between GPCR and G proteins include, on the GPCR side, domains of intracellular loops and C-terminal, which are specific for each family and on the G protein side, essentially the N- et C-terminal domains plus the alpha 4-beta 6 loop. GPCR can adopt several 'active' conformations some of them being found in mutated receptors responsible for pathologies.

Catalytic Domain↗
PubMed2002

G protein-coupled receptor signalling and cross-talk: achieving rapidity and specificity.

Activation of a given type of G protein-coupled receptor (GPCR) triggers a limited set of signalling events in a very rapid and specific manner. The classical paradigm of GPCR signalling was rather linear and sequential. Emerging evidence, however, has revealed that this is only a part of the complex signalling mediated by GPCR. Propagation of GPCR signalling involves cross-regulation of many but specific pathways, including cross-talks between different GPCRs as well as with other signalling pathways. Moreover, it is increasingly apparent that GPCRs can activate both heterotrimeric G protein-dependent and G protein-independent signalling pathways. In this review, we discuss how the signallings initiated by GPCRs achieve rapidity as well as specificity, and how the GPCRs can cross-regulate other specific signalling pathways at the same time. New concepts regarding GPCR signalling have been arising to address this issue, which include multiprotein signalling complex and signalling compartment in microdomain concepts that enable close colocalization or even contact among the proteins engaged in the specific signal transduction. The final outcome of a stimulation of GPCR will thus be the sum of its own specific set of intracellular signalling pathways it regulates.

Animals↗
PubMed2006

G protein-coupled receptors sense fluid shear stress in endothelial cells.

Hemodynamic shear stress stimulates a number of intracellular events that both regulate vessel structure and influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established. Here, we show that mechanical perturbation of the plasma membrane leads to ligand-independent conformational transitions in a G protein-coupled receptor (GPCR). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing agent leads to a significant increase in activity of bradykinin B2 GPCR in endothelial cells. The GPCR conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single endothelial cell under fluid shear stress in real time. We show that this response can be blocked by a B(2)-selective antagonist. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of GPCRs. Therefore, we suggest that GPCRs are involved in mediating primary mechanochemical signal transduction in endothelial cells. We anticipate our experiments to be a starting point for more sophisticated studies of the effects of changes in lipid bilayer environment on GPCR conformational dynamics. Furthermore, because GPCRs are a major target of drug development, a detailed characterization of mechanochemical signaling via the GPCR pathway will be relevant for the development of new antiatherosclerosis drugs.

Animals↗