G Protein-coupled Receptors Biology Research Articles

G Protein-Coupled Receptors in Cancer.

Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

Localization and signaling of GPCRs in lipid rafts.

The understanding of how biological membranes are organized and how they function has evolved. Instead of just serving as a medium in which certain proteins are found, portions of the lipid bilayer have been demonstrated to form specialized platforms that foster the assembly of signaling complexes by providing a microenvironment that is conducive for effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appear to be the best way for drawing conclusions with regards to the distribution and activity of GPCRs in lipid rafts.

Editorial: Orphan GPCRs As Emerging Drug Targets.

Editorial: Orphan GPCRs As Emerging Drug Targets.

PTH receptor-1 signalling-mechanistic insights and therapeutic prospects.

Parathyroid hormone/parathyroid hormone-related protein receptor (PTH/PTHrP type 1 receptor; commonly known as PTHR1) is a family B G-protein-coupled receptor (GPCR) that regulates skeletal development, bone turnover and mineral ion homeostasis. PTHR1 transduces stimuli from PTH and PTHrP into the interior of target cells to promote diverse biochemical responses. Evaluation of the signalling properties of structurally modified PTHR1 ligands has helped to elucidate determinants of receptor function and mechanisms of downstream cellular and physiological responses. Analysis of PTHR1 responses induced by structurally modified ligands suggests that PTHR1 can continue to signal through a G-protein-mediated pathway within endosomes. Such findings challenge the longstanding paradigm in GPCR biology that the receptor is transiently activated at the cell membrane, followed by rapid deactivation and receptor internalization. Evaluation of structurally modified PTHR1 ligands has further led to the identification of ligand analogues that differ from PTH or PTHrP in the type, strength and duration of responses induced at the receptor, cellular and organism levels. These modified ligands, and the biochemical principles revealed through their use, might facilitate an improved understanding of PTHR1 function in vivo and enable the treatment of disorders resulting from defects in PTHR1 signalling. This Review discusses current understanding of PTHR1 modes of action and how these findings might be applied in future therapeutic agents.

Methodological advances: the unsung heroes of the GPCR structural revolution.

G protein-coupled receptors (GPCRs) are intricately involved in a diverse array of physiological processes and pathophysiological conditions. They constitute the largest class of drug target in the human genome, which highlights the importance of understanding the molecular basis of their activation, downstream signalling and regulation. In the past few years, considerable progress has been made in our ability to visualize GPCRs and their signalling complexes at the structural level. This is due to a series of methodological developments, improvements in technology and the use of highly innovative approaches, such as protein engineering, new detergents, lipidic cubic phase-based crystallization and microfocus synchrotron beamlines. These advances suggest that an unprecedented amount of structural information will become available in the field of GPCR biology in the coming years.

From Recombinant Expression to Crystals: A Step-by-Step Guide to GPCR Crystallography.

G protein-coupled receptors (GPCRs) are the primary targets of drugs prescribed for many human pathophysiological conditions such as hypertension, allergies, schizophrenia, asthma, and various types of cancer. High-resolution structure determination of GPCRs has been a key focus area in GPCR biology to understand the basic mechanism of their activation and signaling and to materialize the long-standing dream of structure-based drug design on these versatile receptors. There has been tremendous effort at this front in the past two decades and it has culminated into crystal structures of 27 different receptors so far. The recent progress in crystallization and structure determination of GPCRs has been driven by innovation and cutting-edge developments at every step involved in the process of crystallization. Here, we present a step-by-step description of various steps involved in GPCR crystallization starting from recombinant expression to obtaining diffracting crystals. We also discuss the next frontiers in GPCR biology that are likely to be a primary focus for crystallography efforts in the next decade or so.

Novel Chemical Biology Methods Illuminate the Function of Single G Protein-Coupled Receptors

The superfamily of G Protein-Coupled Receptors (GPCRs) is one of the largest in the human genome. GPCRs are targets for about a quarter of all prescription drugs. Recent progress in the structural biology of GPCRs renews the interest in rational drug design and discovery. Rational design of “functionally selective” drugs or ligands presents a particularly difficult problem. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. However, functional characterization of GPCRs has largely been limited to cell-based high-throughput screening assays. The complexity of the cellular milieu introduces biological variables that make quantitative analysis of structure and dynamics of ligand-receptor complexes that underlie functional selectivity virtually impossible. We are developing single-molecule fluorescence methods to eliminate this biological noise and study such complexes in biochemically-defined systems.[1] Systematic introduction of signaling partners, such as G proteins and arrestins, enables us to study their allosteric effects on ligand-receptor interactions. Recent methodological advances enabled us to express GPCRs with genetically-encoded unnatural amino acids (uaas). We showed that bioorthogonal functional groups on these site-specifically introduced uaas might be used for bioorthogonal reactions. Among a broad spectrum of bioorthogonal reactions, strain-promoted azide-alkyne cycloaddition (SpAAC) with the uaa p-azido-phenylalanine and dibenzocyclooctyne (DIBO) reagents is specific and efficient and presents the best option to obtain stoichiometric receptor-fluorophore conjugates. We designed and built a fully-automatic, multi-color, single-molecule detection fluorescence microscope for long-timescale, multiplexed data acquisition using a multi-well tethered membrane “chip” system. Our convergent technology platform presents a new approach for single-molecule fluorescence studies of the kinetics and dynamics of GPCR signalosomes.[1] T. Huber and T.P. Sakmar. Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling. Chem Biol. 18:1224-1237 (2014)

High-Throughput Screening for Allosteric Modulators of GPCRs.

The continued evolution of our understanding of G protein-coupled receptor (GPCR) signaling has revealed new opportunities for drug discovery. Specifically, biased agonism at GPCRs and allosteric modulation of GPCRs both represent emerging areas of GPCR biology that hold promise for the development of novel GPCR-targeted therapeutics that may provide greater therapeutic efficacy and/or improved side-effect profiles. To obtain initial chemical leads, high-throughput screening (HTS) of a large compound library for the desired activity is often deployed during the early stages of a discovery program. The identification of allosteric modulators, in particular, poses significant challenges for HTS. We describe several HTS protocols designed for the identification of GPCR ligands, with a particular focus on the identification of allosteric modulators.

Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

Deciphering G protein-coupled receptor biology with fluorescence-based methods.

G-protein-coupled receptors (GPCRs) represent the main family of cell surface receptors and are virtually expressed in all eukaryotic cells. Interestingly, a large number of clinically used drugs exert their pharmacological effect via a GPCR, thus it seems crucial to deeply understand the biology of these receptors. The study of GPCR activation and signaling has been classically performed by physiological, biochemical and pharmacological approaches using radioactivity-based tools. However, apart from the potential hazards of radioisotope handling and environmental burden, these approaches have some technical limitations. Therefore, the development of fluorescence-based techniques in general and fluorescence and bioluminescence resonance energy transfer (FRET and BRET) in particular have revolutionized the way to study GPCR functioning both in vitro and in vivo. Indeed, these techniques allow the characterization and visualization of all the individual GPCR signaling steps (i.e. ligand binding, receptor activation, G-protein coupling, G-protein activation, GPCR desensitization) with high temporal and spatial resolution. Here, we review the use and impact of fluorescent-based methodologies on the deciphering of GPCR biology.

GPCRs: Lipid-Dependent Membrane Receptors That Act as Drug Targets

G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and represent major targets in the development of novel drug candidates in all clinical areas. Although there have been some recent leads, structural information on GPCRs is relatively rare due to the difficulty associated with crystallization. A specific reason for this is the intrinsic flexibility displayed by GPCRs, which is necessary for their functional diversity. Since GPCRs are integral membrane proteins, interaction of membrane lipids with them constitutes an important area of research in GPCR biology. In particular, membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. The role of membrane cholesterol in GPCR function is discussed with specific example of theserotonin1Areceptor. Recent results show that GPCRs are characterized with structural motifs that preferentially associate with cholesterol. An emerging and important concept is oligomerization of GPCRs and its role in GPCR function and signaling. The role of membrane cholesterol in GPCR oligomerization is highlighted. Future research in GPCR biology would offer novel insight in basic biology and provide new avenues for drug discovery.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. One structure of a GPCR bound to a G protein was solved, but the structure of a GPCR-arrestin complex has remained unknown despite its central role in GPCR biology. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. The structure reveals that arrestin binding induces large and unexpected conformational changes at both the extracellular and intracellular sides of rhodopsin. Arrestin also undergoes dramatic rearrangements from its inactive well-ordered β-sheet structure into a more flexible molten globule-type state, allowing a snake-like movement of the first 77 arrestin residues that shortens its central crest finger loop by seven residues to accommodate the concave surface of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling, reveals a new paradigm of signal transduction by a molten globule, and demonstrates the extraordinary power of X-ray lasers for advancing the frontiers of structural biology.

Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists

G protein–coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor β-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of β-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.

Portraying G protein-coupled receptors with fluorescent ligands.

The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells.

Application of BRET for studying G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

Regulation ofβ2-Adrenergic Receptor Function by Conformationally Selective Single-Domain Intrabodies

The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.

Applications of molecular replacement to G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

Screening β-Arrestin Recruitment for the Identification of Natural Ligands for Orphan G-Protein–Coupled Receptors

A variety of G-protein–coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated β-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward β-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased β-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter β-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of β-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. β-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through β-arrestin in response to their native ligand remain to be determined.

Molecular signatures of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

Endothelin Receptor Signaling: New Insight Into Its Regulatory Mechanisms

The endothelin (ET) system consists of two G protein coupled–receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca2+ concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca2+ entry and receptor-operated Ca2+ entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca2+ sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1–induced Ca2+ mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca2+ signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.