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PubMed1999

Regulation of tyrosine kinase cascades by G-protein-coupled receptors.

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.

Animals↗
PubMed2001

Crystal structure of rhodopsin: implications for vision and beyond.

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.

Animals↗
PubMed2000

G-protein-coupled receptors function as oligomers in vivo.

Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non-functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild-type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation.

Endocytosis↗
PubMed2002

Lessons from constitutively active mutants of G protein-coupled receptors.

In the past decade, the concept of constitutive activity has profoundly modified our understanding of G protein-coupled-receptors (GPCRs). Here, we review the contribution of constitutively active mutants (CAMs) to our understanding of three aspects of GPCR physiopathology: (1) GPCR activation is a complex mechanism involving both the release of inactive state conformational constraints, mimicked by most CAMs, and the creation of new interactions that stabilize the active state and are mimicked by a restricted set of CAMs; (2) GPCR phosphorylation, internalization and desensitization processes are activated by receptor conformations, which partly overlap those activating G protein; (3) natural CAMs, mostly affecting GPCRs of the endocrine system, are found in several hereditary and acquired diseases, including cancers. One major remaining question is how CAMs recapitulate the different structural modifications of the agonist-induced active conformation(s) of the wild-type receptor. This characterization is a prerequisite for further use of CAMs as ligand-free models of active GPCRs in structural, cellular and physiological studies.

Amino Acid Sequence↗
PubMed2002

Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes.

The existence of dimers and higher oligomers of G-protein-coupled receptors (GPCRs) has been frequently reported using strategies based on coimmunoprecipitation or Western blot assays. These methods rely on highly artificial systems with overexpressed receptors, resulting in conflicting observations on the question of whether GPCR dimers are preformed or are formed in response to agonist treatment. Fluorescence resonance energy transfer (FRET) microscopy is a superior and less perturbing technique which can be performed on selected cell regions, e.g., plasma membrane of intact cells with a sensitivity high enough to allow study under physiological levels of receptor expression. Here we describe the application of photobleaching (pb) FRET microscopy for investigating ligand-dependent oligomerization of somatostatin receptors. Procedures for the introduction of suitable donor-acceptor fluorophores in a given GPCR are described. The competitive nature of FRET and photobleaching is exploited to enable the indirect measurement of FRET via its effect on donor photobleaching lifetimes on a pixel-by-pixel basis. The method allows enhanced resolution between 10 and 100A and represents a sensitive and specific biophysical tool for characterizing the assembly and regulation of GPCR oligomers on the cell surface.

Animals↗
PubMed2005

Identifying G-protein coupled receptors using weighted Levenshtein distance and the nearest neighbor method.

G-protein coupled receptors (GPCRs) are a class of seven-helix transmembrane proteins that have been used in bioinformatics as the targets to facilitate drug discovery for human diseases. Although thousands of GPCR sequences have been collected, the ligand specificity of many GPCRs are still unknown and only one crystal structure of the rhodopsin-like family has been solved. Therefore, identifying GPCR types only from sequence data has become an important research issue. In this study, a novel technique for identifying GPCR types based on the weighted Levenshtein distance between two receptor sequences and the nearest neighbor method (NNM) is introduced, which can deal with receptor sequences with different lengths directly. In our experiments for classifying four classes (acetylcholine, adrenoceptor, dopamine, and serotonin) of the rhodopsin-like family of GPCRs, the error rates from the leave-one-out procedure and the leave-half-out procedure are 0.62% and 1.24%, respectively. These results are prior to those of the covariant discriminant algorithm, the support vector machine method, and the NNM with Euclidean distance.

Algorithms↗
PubMed2002

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.

Affinity Labels↗
PubMed2005

Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [(35)S]GTPgammaS binding assays of the 5HT(1A) and EDG(1) GPCRs confirmed that they were properly folded and functional. 5HT(1A)R was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.

Animals↗
PubMed1999

Multiple activation steps of the N-formyl peptide receptor.

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.

Animals↗
PubMedDate not supplied

Optimization of the human adenosine A2a receptor yields in Saccharomyces cerevisiae.

G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A(2)a receptor (A(2)aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A(2)aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD(600)) and per cell A(2)aR expression levels. For these studies, the A(2)aR expression levels were determined using a previously described A(2)aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times ( approximately 60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A(2)aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points ( approximately 60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization.

Biotechnology↗
PubMedDate not supplied

An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

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. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

Amino Acid Sequence↗
PubMed2002

Membrane protein microarrays.

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.

Fluorescence↗
PubMed2001

Dimerization of G-protein-coupled receptors.

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

Amino Acid Sequence↗
PubMed2005

Virtual screening of biogenic amine-binding G-protein coupled receptors: comparative evaluation of protein- and ligand-based virtual screening protocols.

In this paper, we compare protein- and ligand-based virtual screening techniques for identifying the ligands of four biogenic amine-binding G-protein coupled receptors (GPCRs). For the screening of the virtual compound libraries, we used (1) molecular docking into GPCR homology models, (2) ligand-based pharmacophore and Feature Tree models, (3) three-dimensional (3D)-similarity searches, and (4) statistical methods [partial least squares (PLS) and partial least squares discriminant analysis (PLS-DA) models] based on two-dimensional (2D) molecular descriptors. The comparison of the different methods in retrieving known antagonists from virtual libraries shows that in our study the ligand-based pharmacophore-, Feature Tree-, and 2D quantitative structure-activity relationship (QSAR)-based screening techniques provide enrichment factors that are higher than those provided by molecular docking into the GPCR homology models. Nevertheless, the hit rates achieved when docking with GOLD and ranking the ligands with GoldScore (up to 60% among the top-ranked 1% of the screened databases) are still satisfying. These results suggest that docking into GPCR homology models can be a useful approach for lead finding by virtual screening when either little or no information about the active ligands is available.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1999

Visualisation and integration of G protein-coupled receptor related information help the modelling: description and applications of the Viseur program.

G Protein-Coupled Receptors (GPCRs) constitute a superfamily of receptors that forms an important therapeutic target. The number of known GPCR sequences and related information increases rapidly. For these reasons, we are developing the Viseur program to integrate the available information related to GPCRs. The Viseur program allows one to interactively visualise and/or modify the sequences, transmembrane areas, alignments, models and results of mutagenesis experiments in an integrated environment. This integration increases the ease of modelling GPCRs: visualisation and manipulation improvements enable easier databank interrogation and interpretation. Unique program features include: (i) automatic construction of 'Snake-like' diagrams or hyperlinked GPCR molecular models to HTML or VRML and (ii) automatic access to a mutagenesis data server through the Internet. The novel algorithms or methods involved are presented, followed by the overall complementary features of the program. Finally, we present two applications of the program: (i) an automatic construction of GPCR snake-like diagrams for the GPCRDB WWW server, and (ii) a preparation of the modelling of the 5HT receptor subtypes. The interest of the direct access to mutagenesis results through an alignment and a molecular model are discussed. The Viseur program, which runs on SGI workstations, is freely available and can be used for preparing the modelling of integral membrane proteins or as an alignment editor tool.

Algorithms↗
PubMed2004

Detection of pairwise residue proximity by covariation analysis for 3D-structure prediction of G-protein-coupled receptors.

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.

Animals↗
PubMed2002

Ligand based structural studies of the CB1 cannabinoid receptor.

The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.

Amino Acids↗
PubMed1999

Ocular albinism: evidence for a defect in an intracellular signal transduction system.

G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.

Albinism, Ocular↗