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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.

G protein-coupled receptors (GPCRs) are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that GPCRs form dimers and various studies have shown that dimerization is necessary for receptor maturation, signaling and trafficking. However, the physiological implications of dimerization in vivo have not been well explored since detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific GPCR dimers. Such antibodies could be used as tools for characterization of heteromer-specific function, as reagents for their purification, tissue localization and regulation in vivo and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for GPCR heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of GPCR dimer pharmacology.

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.

For years conventional drug design at G-protein coupled receptors (GPCRs) has mainly focused on the inhibition of a single receptor at a usually well-defined ligand-binding site. The recent discovery of more and more physiologically relevant GPCR dimers/oligomers suggests that selectively targeting these complexes or designing small molecules that inhibit receptor-receptor interactions might provide new opportunities for novel drug discovery. To uncover the fundamental mechanisms and dynamics governing GPCR dimerization/oligomerization, it is crucial to understand the dynamic process of receptor-receptor association, and to identify regions that are suitable for selective drug binding. This minireview highlights current progress in the development of increasingly accurate dynamic molecular models of GPCR oligomers based on structural, biochemical, and biophysical information that has recently appeared in the literature. In view of this new information, there has never been a more exciting time for computational research into GPCRs than at present. Information-driven modern molecular models of GPCR complexes are expected to efficiently guide the rational design of GPCR oligomer-specific drugs, possibly allowing researchers to reach for the high-hanging fruits in GPCR drug discovery, i.e. more potent and selective drugs for efficient therapeutic interventions.

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.

The modulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes the single most important therapeutic target in medicine. Drugs acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two-state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and “texture” than previously appreciated. Signal diversity arises from numerous factors, among which are the ability of receptors to adopt multiple “active” states with different effector-coupling profiles; the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking; the dissociation of receptor “activation” from desensitization and internalization; and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confer signal preorganization, efficiency, and specificity. In this context, the concept of agonist-selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of active receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands possessing increased specific efficacy and attenuated side effects may become the standard mode of drug development.