Quantifying agonist activity at G protein-coupled receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (τ). The estimate of τK(obs) of one agonist, divided by that of another, is a relative measure of K(b) (RA(i)). For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τ(sys)). In this case, the K(b) value of an agonist is equivalent to τK(obs)/τ(sys). Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.     

Continued discovery of ligands for G protein-coupled receptors

G protein-coupled receptors are under intense scrutiny as potential targets of drug research, which stems mostly from the sheer size and diversity of this receptor family as well as the recognized high levels of specificity and sensitivity attainable by drugs targeting these receptors. The continued discovery of genes encoding G protein-coupled receptors has provided an extensive reserve of potential therapeutic targets. However, testing experimental therapeutic agents at these receptors requires a high degree of receptor characterization, beginning with the identity of an endogenous ligand. Often, low levels of sequence identity of a newly identified receptor to previously characterized receptors preclude the prompt identification of a ligand. In such cases, innovative techniques commonly referred to as reverse pharmacology have been employed to ascertain the ligand's identity for these "orphan" receptors. To date over 30 endogenous ligands, both novel and previously known, have been paired with orphan G protein-coupled receptors. Here, we briefly summarize the recent identification of neuropeptides W and B and carboxylic acid anions for their respective receptors GPR7, GPR8 and GPR40, GPR41, GPR43.     

Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors

We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha16. We now show that, compared with Galpha16, the use of chimeric Galphaq subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect Gi-coupled GPCRs into a Gq-type signal transduction pathway. We coexpressed human Gi-coupled GPCRs, G protein alpha subunits (either a chimeric Galphaq or Galpha16), and the calcium-sensitive reporter protein aequorin in Sf9 cells using a nonlytic protein expression system, and measured agonist-induced intracellular calcium flux using a luminometer. Three of the GPCRs (serotonin 1A, 1D, and dopamine D2) were functionally redirected into a Gq-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha16. We determined agonist concentration-response relationships for all three receptors, which yielded EC50 values comparable with those achieved in mammalian cell-based assay systems. However, three other Gi-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha16. Possible reasons and solutions for this result are discussed.     

Identification of surrogate ligands for orphan G protein-coupled receptors

We prepared fusion proteins with an alpha subunit of G protein Gi (Gi1alpha) of 26 orphan G protein-coupled receptors (GPCRs) and with Gsalpha of 10 orphan GPCRs, most of which had been identified from the human genome previously [FEBS Lett 520 (2002) 97]. Ligands for these fusion proteins were screened from a library consisting of approximately 1000 authentic compounds by measuring their effect on [35S]GTPgammaS binding to membrane preparations of insect Sf9 cells expressing these fusion proteins. Eleven compounds were found to act as surrogate agonists for a GPCR-Gsalpha and four GPCR-Gialpha fusion proteins, a compound as an inverse agonist for two GPCR-Gsalpha fusion proteins, and a compound as an endogenous agonist for a GPCR-Gialpha fusion protein.     

Fluorescence polarization assays for high throughput screening of G protein-coupled receptors

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to [(125)I]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 microM. These attributes suggest that accurate assessment of drug binding can be obtained.     

Comparative analysis of the heptahelical transmembrane bundles of G protein-coupled receptors

Background

G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices.

Methodology/Principal Findings

We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands.

Conclusions/Significance

Systematic analysis described here would be valuable for understanding of the rearrangement of seven helices which depends on the ligand specificity and activation state of the receptors.     

Biased agonism at G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of approved therapeutic targets. Ligands stimulating these receptors specifically activate multiple signalling pathways that induce not only the desired therapeutic response, but sometimes untolerated side effects that limit their clinical use. The diversity in signalling induced by each ligand could be considered a viable path for improving this situation. Biased agonism, which offers the promise of identifying pathway-selective drugs has been proposed as a means to exploit this opportunity. However, identifying biased agonists is not an easy process and quantifying ligand bias for a given signalling pathway requires careful consideration and control of several confounding factors. To date, the molecular mechanisms of biased signalling remain unclear and known theories that constitute our understanding of the mechanisms underlying therapeutic and side effects are still being challenged, making the strategy of selecting promising potential drugs more difficult. This special issue summarizes the latest advances in the discovery and optimization of biased ligands for different GPCRs. It also focuses on identifying novel insights into the field of biased agonism, while at the same time, highlighting the conceptual and experimental limitations of that concept for drug discovery. This aims to broaden our understanding of the signalling induced by the various identified biased agonists and provide perspectives that could straighten our path towards the development of more effective and tolerable therapeutics.     

Therapeutic antibodies directed at G protein-coupled receptors

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.Key words: G protein-coupled receptor, transmembrane spanning domain, chemokine receptor, extracellular domain, extracellular loop     

Therapeutic antibodies directed at G protein-coupled receptors

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 over-expressing 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.     

Cell-free production of G protein-coupled receptors for functional and structural studies

G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24 h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.     

Identification and characterization of two G protein-coupled receptors for neuropeptide FF

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.     

Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors

G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein. The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics. In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods. BLAST and FastA offer broad brush strokes. Motif-based search methods add the fine detail. Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding. Together, these different views provide a more informative and a more detailed picture of GPCR structure and function. Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow.     

Conformational changes of G protein-coupled receptors during their activation by agonist binding

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high-resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high-resolution x-ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three-dimensional GPCR structure can be included into pharmaceutical drug design programs.     

G protein-coupled receptors.

The diversity of the G protein-coupled receptor superfamily is now being realised with the molecular cloning of DNA encoding many new receptors and receptor subfamilies. The existing pharmacological definitions of receptor subtypes have been extended dramatically with identification of additional subtypes at the molecular level. Functional analysis of cloned receptors by expression in heterologous cell types has demonstrated that individual receptor subtypes can couple to a variety of different effector systems.     

Lysophospholipid G protein-coupled receptors

The many biological responses documented for lysophospholipids that include lysophosphatidic acid and sphingosine 1-phosphate can be mechanistically attributed to signaling through specific G protein-coupled receptors. At least nine receptors have now been identified, and the total number is likely to be larger. In this brief review, we note cogent features of lysophospholipid receptors, including the current nomenclature, signaling properties, development of agonists and antagonists, and physiological functions.     

Innovative functional cAMP assay for studying G protein-coupled receptors: application to the pharmacological characterization of GPR17

In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC₅₀ or IC₅₀ values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [³⁵S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G_αi.     

Downregulation of G protein-coupled receptors

Major advances have been made in understanding mechanisms mediating downregulation of G protein-coupled receptors. Recent studies emphasize the role of multiple proteolytic mechanisms in downregulation. A specific mechanism of downregulation, mediated by endocytosis of receptors via clathrin-coated pits followed by sorting to lysosomes, has been examined in detail. Specific protein interactions that control the specificity of G-protein-coupled receptor trafficking in this pathway are beginning to be elucidated.     

Comparative genomic analysis and evolution of family-B G protein-coupled receptors from six model insect species

Family-B G protein-coupled receptors (GPCR-Bs) play vital roles in many biological processes, including growth, development and reproduction. However, the evolution and function of GPCR-Bs have been poorly understood in insects.