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1.  Modern Homology Modeling of G-Protein Coupled Receptors: Which Structural Template to Use? 
Journal of medicinal chemistry  2009;52(16):5207-5216.
The recent availability in the literature of new crystal structures of inactive G-protein coupled receptors (GPCRs) prompted us to study the extent to which these crystal structures constitute an advantage over the former prototypic rhodopsin template for homology modeling of the transmembrane (TM) region of human class A GPCRs. Our results suggest that better templates than those currently available are required by the majority of these GPCRs to generate homology models that are accurate enough for simple virtual screening aimed at computer-aided drug discovery. Thus, we investigated: 1) which class A GPCRs would have the highest impact as potential templates for homology modeling of other GPCRs, if their structures were solved; and 2) the extent to which multiple-template homology modeling (using all currently available GPCR crystal structures) provides an improvement over single-template homology modeling, as evaluated by the accuracy of rigid protein-flexible ligand docking on these models.
doi:10.1021/jm9005252
PMCID: PMC2891345  PMID: 19627087
2.  G protein-coupled receptor drug discovery: Implications from the crystal structure of rhodopsin 
G protein-coupled receptors (GPCRs) are a functionally diverse group of membrane proteins that play a critical role in signal transduction. Because of the lack of a high-resolution structure, the heptahelical transmembrane bundle within the N-terminal extracellular and C-terminal intracellular region of these receptors has initially been modeled based on the high-resolution structure of bacterial retinal-binding protein, bacteriorhodopsin. However, the low-resolution structure of rhodopsin, a prototypical GPCR, revealed that there is a minor relationship between GPCRs and bacteriorhodopsins. The high-resolution crystal structure of the rhodopsin ground state and further refinements of the model provide the first structural information about the entire organization of the polypeptide chain and post-translational moieties. These studies provide a structural template for Family 1 GPCRs that has the potential to significantly improve structure-based approaches to GPCR drug discovery.
PMCID: PMC1383658  PMID: 12825452
Agonist; antagonist; drug discovery and design; G protein; G protein-coupled receptors (GPCRs); rhodopsin; signal transduction; β2R β2-adrenergic receptor; EPI epinephrine; FVS fuzzy virtual screening; GPCR G protein-coupled receptors SAR structure-activity relationship; SCAM substituted Cys accessibility method; TM transmembrane
3.  Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies 
Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function.
doi:10.1098/rsif.2012.0846
PMCID: PMC3565703  PMID: 23235263
calcitonin gene-related peptide; GCR1; molecular dynamics; family B G-protein-coupled receptor; family A G-protein-coupled receptor; motifs
4.  Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface 
PLoS ONE  2012;7(5):e37136.
G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.
doi:10.1371/journal.pone.0037136
PMCID: PMC3356411  PMID: 22623985
5.  Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors 
A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.
doi:10.1016/j.bmcl.2006.07.015
PMCID: PMC1810397  PMID: 16931008
Melanocortins; Peptide mimetics; GPCRs
6.  GLIDA: GPCR-ligand database for chemical genomic drug discovery 
Nucleic Acids Research  2005;34(Database issue):D673-D677.
G-protein coupled receptors (GPCRs) represent one of the most important families of drug targets in pharmaceutical development. GPCR-LIgand DAtabase (GLIDA) is a novel public GPCR-related chemical genomic database that is primarily focused on the correlation of information between GPCRs and their ligands. It provides correlation data between GPCRs and their ligands, along with chemical information on the ligands, as well as access information to the various web databases regarding GPCRs. These data are connected with each other in a relational database, allowing users in the field of GPCR-related drug discovery to easily retrieve such information from either biological or chemical starting points. GLIDA includes structure similarity search functions for the GPCRs and for their ligands. Thus, GLIDA can provide correlation maps linking the searched homologous GPCRs (or ligands) with their ligands (or GPCRs). By analyzing the correlation patterns between GPCRs and ligands, we can gain more detailed knowledge about their interactions and improve drug design efforts by focusing on inferred candidates for GPCR-specific drugs. GLIDA is publicly available at . We hope that it will prove very useful for chemical genomic research and GPCR-related drug discovery.
doi:10.1093/nar/gkj028
PMCID: PMC1347391  PMID: 16381956
7.  Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 
PLoS ONE  2011;6(8):e23076.
G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.
doi:10.1371/journal.pone.0023076
PMCID: PMC3154916  PMID: 21853070
8.  Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster 
The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation.
doi:10.3389/fendo.2012.00093
PMCID: PMC3414713  PMID: 22908006
invertebrate neuropeptides; G-protein coupled receptor; insects; nematodes; Caenorhabditis elegans; Drosophila melanogaster
9.  Exploratory Studies on Development of the Chemokine Receptor CXCR4 Antagonists Toward Downsizing 
Seven transmembrane (7TM) G-protein-coupled receptor (GPCR) families are important targets for drug discovery, and specific antagonists for GPCR can accelerate research in the field of medicinal chemistry. The chemokine receptor CXCR4 is a GPCR that possesses a unique ligand CXCL12/stromal cell-derived factor-1 (SDF-1). The interaction between CXCL12 and CXCR4 is essential for the migration of progenitor cells during embryonic development of the cardiovascular, hemopoietic and central nervous systems, and also involved in several intractable disease processes, including HIV infection, cancer cell metastasis, progression of acute and chronic leukemias, rheumatoid arthritis and pulmonary fibrosis. Thus, CXCR4 may be an important therapeutic target in all of these diseases, and various CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogs, and downsized cyclic pentapeptides have been developed by us as potent CXCR4 antagonists. This article describes the development of a number of specific CXCR4 antagonists in our laboratory, including downsizing.
PMCID: PMC2746577  PMID: 19787093
cancer metastasis; chemokine receptor; CXCR4 antagonist; downsizing; HIV infection; rheumatoid arthritis
10.  Homology Modeling of Class A G Protein-Coupled Receptors 
G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that hold great pharmaceutical interest. Since experimentally elucidated structures are available only for a very limited number of receptors, homology modeling has become a widespread technique for the construction of GPCR models intended to study the structure-function relationships of the receptors and aid the discovery and development of ligands capable of modulating their activity. Through this chapter, various aspects involved in the constructions of homology models of the serpentine domain of the largest class of GPCRs, known as class A or rhodopsin family, are illustrated. In particular, the chapter provides suggestions, guidelines and critical thoughts on some of the most crucial aspect of GPCR modeling, including: collection of candidate templates and a structure-based alignment of their sequences; identification and alignment of the transmembrane helices of the query receptor to the corresponding domains of the candidate templates; selection of one or more templates receptor; election of homology or de novo modeling for the construction of specific extracellular and intracellular domains; construction of the three-dimensional models, with special consideration to extracellular regions, disulfide bridges, and interhelical cavity; validation of the models through controlled virtual screening experiments.
doi:10.1007/978-1-61779-588-6_11
PMCID: PMC3354613  PMID: 22323225
G protein-coupled receptors (GPCRs); membrane spanning helices; extracellular loops; homology modeling; de novo modeling; multiple sequence alignment; model validation; controlled virtual screening
11.  Therapeutic antibodies directed at G protein-coupled receptors 
mAbs  2010;2(6):594-606.
G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in overexpressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.
doi:10.4161/mabs.2.6.13420
PMCID: PMC3011214  PMID: 20864805
G protein-coupled receptor; transmembrane spanning domain; chemokine receptor; extracellular domain; extracellular loop
12.  Evolutionary relationships among G protein-coupled receptors using a clustered database approach 
AAPS PharmSci  2001;3(2):25-42.
Guanine nucleotide-binding proteincoupled receptors (GPCRs) comprise large and diverse gene families in fungi, plants, and the animal kingdom. GPCRs appear to share a common structure with 7 transmembrane segments, but sequence similarity is minimal among the most distant GPCRs. To reevaluate the question of evolutionary relationships among the disparate GPCR families, this study takes advantage of the dramatically increased number of cloned GPCRs. Sequences were selected from the National Center for Biotechnology Information (NCBI) nonredundant peptide database using iterative BLAST (Basic Local Alignment Search Tool) searches to yield a database of ∼1700 GPCRs and unrelated membrane proteins as controls, divided into 34 distinet clusters. For each cluster, separate position-specific matrices were established to optimize sequence comparisons among GPCRs. This approach resulted in significant alignments between distant GPCR families, including receptors for the biogenic amine/peptide, VIP/secretin, cAMP, STE3/MAP3 fungal pheromones, latrophilin, developmental receptors frizzled and smoothened, as well as the more distant metabotrobic glutamate receptors, the STE2/MAM2 fungal pheromone receptors, and GPR1, a fungal glucose receptor. On the other hand, alignment scores between these recognized GPCR clades with p40 (putative GPCR) and pml (putative GPCR), as well as bacteriorhodopsins, failed to support a finding of homology. This study provides a refined view of GPCR ancestry and serves as a reference database with hyperlinks to other sources. Moreover, it may facilitate database annotation and the assignment of orphan receptors to GPCR families.
doi:10.1208/ps030212
PMCID: PMC2779559  PMID: 11741263
13.  Functional expression and characterization of the C. elegans G-protein-coupled FLP-2 Receptor (T19F4.1) in mammalian cells and yeast 
Graphical abstract
Highlights
► Nematode neuropeptide G protein-coupled receptors are good targets for anthelmintic discovery. ► Neuropeptides encoded on the Caenorhabditis elegans flp-2 gene are bioactive in parasite assays. ► The receptor for flp-2 peptides has been functionally expressed in mammalian cells and yeast. ► The recombinant yeast expressing this receptor is the focus of an ongoing drug discovery effort.
Profound neuropeptide diversity characterizes the nematode nervous system, but it has proven challenging to match neuropeptide G protein-coupled receptors (GPCR) with their cognate ligands in heterologous systems. We have expressed the Caenorhabditis elegans GPCR encoded in the locus T19F4.1, previously matched with FMRFamide-like peptides encoded on the flp-2 precursor gene, in mammalian cells and in the yeast Saccharomyces cerevisiae. Pharmacological characterization revealed that the receptor is potently activated by flp-2 peptides in CHO cells (∼10 nM EC50) and in yeast (∼100 nM EC50), signaling through a Gqα pathway in each system. The yeast GPCR expression system provides a robust assay for screening for agonists of the flp-2 receptor and is the target of an ongoing high-throughput screening exercise.
doi:10.1016/j.ijpddr.2012.10.002
PMCID: PMC3862401  PMID: 24533288
flp, FMRFamide-like protein gene; FLP, FMRFamide-like peptide; FLP2-R, receptor for peptides encoded on flp-2; FLIPR, fluorescence imaging plate reader; GPCR, G protein-coupled receptor; nlp, neuropeptide-like protein gene; FLP; Caenorhabditis elegans; flp2; Neuropeptide
14.  The Crystallographic Model of Rhodopsin and Its Use in Studies of Other G Protein–Coupled Receptors 
G protein–coupled receptors (GPCRs) are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways activating cellular processes. Rhodopsin is a GPCR found in rod cells in retina where it functions as a photopigment. Its molecular structure is known from cryo-electron microscopic and X-ray crystallographic studies, and this has reshaped many structure/function questions important in vision science. In addition, this first GPCR structure has provided a structural template for studies of other GPCRs, including many known drug targets. After presenting an overview of the major structural elements of rhodopsin, recent literature covering the use of the rhodopsin structure in analyzing other GPCRs will be summarized. Use of the rhodopsin structural model to understand the structure and function of other GPCRs provides strong evidence validating the structural model.
doi:10.1146/annurev.biophys.32.110601.142520
PMCID: PMC1351250  PMID: 12574068
transmembrane protein; signal transduction; homology models; vision; phototransduction
15.  GOMoDo: A GPCRs Online Modeling and Docking Webserver 
PLoS ONE  2013;8(9):e74092.
G-protein coupled receptors (GPCRs) are a superfamily of cell signaling membrane proteins that include >750 members in the human genome alone. They are the largest family of drug targets. The vast diversity and relevance of GPCRs contrasts with the paucity of structures available: only 21 unique GPCR structures have been experimentally determined as of the beginning of 2013. User-friendly modeling and small molecule docking tools are thus in great demand. While both GPCR structural predictions and docking servers exist separately, with GOMoDo (GPCR Online Modeling and Docking), we provide a web server to seamlessly model GPCR structures and dock ligands to the models in a single consistent pipeline. GOMoDo can automatically perform template choice, homology modeling and either blind or information-driven docking by combining together proven, state of the art bioinformatic tools. The web server gives the user the possibility of guiding the whole procedure. The GOMoDo server is freely accessible at http://molsim.sci.univr.it/gomodo.
doi:10.1371/journal.pone.0074092
PMCID: PMC3772745  PMID: 24058518
16.  Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System 
PLoS ONE  2013;8(6):e66793.
Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.
doi:10.1371/journal.pone.0066793
PMCID: PMC3689660  PMID: 23805278
17.  Deorphanization of Novel Peptides and Their Receptors 
The AAPS Journal  2010;12(3):378-384.
Peptide hormones and neuropeptides play important roles in endocrine and neural signaling, often using G protein-coupled receptor (GPCR)-mediated signaling pathways. However, the rate of novel peptide discovery has slowed dramatically in recent years. Genomic sequencing efforts have yielded a large number of cDNA sequences that potentially encode novel candidate peptide precursors, as well as hundreds of orphan GPCRs with no known cognate ligands. The complexity of peptide signaling is further highlighted by the requirement for specific posttranslational processing steps, and these must be accomplished in vitro prior to testing newly discovered peptide precursor candidates in receptor assays. In this review, we present historic as well as current approaches to peptide discovery and GPCR deorphanization. We conclude that parallel and combinatorial discovery methods are likely to represent the most fruitful avenues for both peptide discovery as well as for matching the remaining GPCRs with their peptide ligands.
doi:10.1208/s12248-010-9198-9
PMCID: PMC2895435  PMID: 20446073
deorphanization; GPCR; peptide hormones; proprotein convertases; screening
18.  Structure-based drug screening for G protein-coupled receptors 
G protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past four years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.
doi:10.1016/j.tips.2012.03.007
PMCID: PMC3523194  PMID: 22503476
19.  Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry 
Amino Acids  2012;44(2):581-595.
Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-012-1376-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00726-012-1376-x
PMCID: PMC3549257  PMID: 22890611
Viola ignobilis; Circular; Cystine-knot; Oxidative folding; Vigno; Peptidomics
20.  The G protein-coupled receptor subset of the dog genome is more similar to that in humans than rodents 
BMC Genomics  2009;10:24.
Background
The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species.
Results
We searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog.
Conclusion
The repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.
doi:10.1186/1471-2164-10-24
PMCID: PMC2651185  PMID: 19146662
21.  Structure Modeling of All Identified G Protein–Coupled Receptors in the Human Genome 
PLoS Computational Biology  2006;2(2):e13.
G protein–coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global Cα root-mean-squared deviation from native of 4.6 Å, with a root-mean-squared deviation in the transmembrane helix region of 2.1 Å. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness of the in silico models for GPCR functional analysis. All predicted GPCR models are freely available for noncommercial users on our Web site (http://www.bioinformatics.buffalo.edu/GPCR).
Synopsis
G protein–coupled receptors (GPCRs) are a large superfamily of integral membrane proteins that transduce signals across the cell membrane. Because of the breadth and importance of the physiological roles undertaken by the GPCR family, many of its members are important pharmacological targets. Although the knowledge of a protein's native structure can provide important insight into understanding its function and for the design of new drugs, the experimental determination of the three-dimensional structure of GPCR membrane proteins has proved to be very difficult. This is demonstrated by the fact that there is only one solved GPCR structure (from bovine rhodopsin) deposited in the Protein Data Bank library. In contrast, there are no human GPCR structures in the Protein Data Bank. To address the need for the tertiary structures of human GPCRs, using just sequence information, the authors use a newly developed threading-assembly-refinement method to generate models for all 907 registered GPCRs in the human genome. About 820 GPCRs are anticipated to have correct topology and transmembrane helix arrangement. A subset of the resulting models is validated by comparison with mutagenesis experimental data, and consistent agreement is demonstrated.
doi:10.1371/journal.pcbi.0020013
PMCID: PMC1364505  PMID: 16485037
22.  Structure Modeling of All Identified G Protein–Coupled Receptors in the Human Genome 
PLoS Computational Biology  2006;2(2):e13.
G protein–coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global Cα root-mean-squared deviation from native of 4.6 Å, with a root-mean-squared deviation in the transmembrane helix region of 2.1 Å. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness of the in silico models for GPCR functional analysis. All predicted GPCR models are freely available for noncommercial users on our Web site (http://www.bioinformatics.buffalo.edu/GPCR).
Synopsis
G protein–coupled receptors (GPCRs) are a large superfamily of integral membrane proteins that transduce signals across the cell membrane. Because of the breadth and importance of the physiological roles undertaken by the GPCR family, many of its members are important pharmacological targets. Although the knowledge of a protein's native structure can provide important insight into understanding its function and for the design of new drugs, the experimental determination of the three-dimensional structure of GPCR membrane proteins has proved to be very difficult. This is demonstrated by the fact that there is only one solved GPCR structure (from bovine rhodopsin) deposited in the Protein Data Bank library. In contrast, there are no human GPCR structures in the Protein Data Bank. To address the need for the tertiary structures of human GPCRs, using just sequence information, the authors use a newly developed threading-assembly-refinement method to generate models for all 907 registered GPCRs in the human genome. About 820 GPCRs are anticipated to have correct topology and transmembrane helix arrangement. A subset of the resulting models is validated by comparison with mutagenesis experimental data, and consistent agreement is demonstrated.
doi:10.1371/journal.pcbi.0020013
PMCID: PMC1364505  PMID: 16485037
23.  Intercellular Lipid Mediators and GPCR Drug Discovery 
Biomolecules & Therapeutics  2013;21(6):411-422.
G-protein-coupled receptors (GPCR) are the largest superfamily of receptors responsible for signaling between cells and tissues, and because they play important physiological roles in homeostasis, they are major drug targets. New technologies have been developed for the identification of new ligands, new GPCR functions, and for drug discovery purposes. In particular, intercellular lipid mediators, such as, lysophosphatidic acid and sphingosine 1-phosphate have attracted much attention for drug discovery and this has resulted in the development of fingolimod (FTY-720) and AM095. The discovery of new intercellular lipid mediators and their GPCRs are discussed from the perspective of drug development. Lipid GPCRs for lysophospholipids, including lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylcholine, free fatty acids, fatty acid derivatives, and other lipid mediators are reviewed.
doi:10.4062/biomolther.2013.080
PMCID: PMC3879912  PMID: 24404331
Lipid mediator; GPCR; Lipid; Lysophospholipid; Fatty acid; Drug discovery
24.  Discovery of New GPCR biology – One Receptor Structure at a Time 
Summary
G protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome. They transmit an exogenous signal to the intracellular second messenger cascade by ligand induced conformational changes within a common seven transmembrane helical bundle. Due to their critical role in biology and drug discovery, tremendous effort has been made over the past several decades to understand the mechanism of signal transduction through the cell membrane. Within the last year we have witnessed a relative explosion in the amount of structural information available for the GPCR family with two new structures of opsin in the presence and absence of transducin peptide, four new structures of β-adrenergic receptors and a recent structure of the human adenosine A2A receptor. The new biological insight being gained such as the highly divergent extracellular loops and areas of structural convergence within the transmembrane helices, allows us to chart a course for further investigation into this important class of membrane proteins.
doi:10.1016/j.str.2008.12.003
PMCID: PMC2813843  PMID: 19141277
25.  Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders 
Despite G-protein-coupled receptors (GPCRs) being among the most fruitful targets for marketed drugs, intense discovery efforts for several GPCR subtypes have failed to deliver selective drug candidates. Historically, drug discovery programmes for GPCR ligands have been dominated by efforts to develop agonists and antagonists that act at orthosteric sites for endogenous ligands. However, in recent years, there have been tremendous advances in the discovery of novel ligands for GPCRs that act at allosteric sites to regulate receptor function. These compounds provide high selectivity, novel modes of efficacy and may lead to novel therapeutic agents for the treatment of multiple psychiatric and neurological human disorders.
doi:10.1038/nrd2760
PMCID: PMC2907734  PMID: 19116626

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