Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins

GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of β-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs.     

Mrg受体家族的研究进展

Mrg(mas related gene)受体家族是新发现的G蛋白偶联受体家族,特异性分布于感觉传入神经元上。研究发现Mrg受体家族参与了痛觉、免疫等生理病理过程;Mrg受体的内源性配体的寻找和构效关系研究对揭示此受体系统的生理学角色有重要意义;同时针对Mrg受体及其配体的药理学研究对于相关疾病的治疗提供了一个新靶点。     

The G-protein coupled receptor family: actors with many faces

G-protein coupled receptors (GPCRs) comprise the largest family of proteins in our body, which have many important physiological functions and are implicated in the pathophysiology of many serious diseases. GPCRs therefore are significant targets in pharmaceutical research. GPCRs share the common architecture of seven plasma membrane-spanning segments connected to each other with three extracellular and three intracellular loops. In addition, GPCRs contain an extracellular N-terminal region and an intracellular C-terminal tail. GPCRs could stimulate different intracellular G-proteins (internal stimuli) and signaling pathways after their interaction with different ligands (external stimuli). The exceptional functional plasticity of GPCRs could be attributed to their inherent dynamic nature to adopt different active conformations, which are stabilized differentially by different stimuli as well as by several mutations. This review describes the structural changes of GPCRs associated with their activation. Understanding the dynamic nature of GPCRs could potentially contribute in the development of future structure-based approaches to design new receptor-specific, signaling-selective ligands, which will enrich the pharmaceutical armamentarium against various diseases.     

Orphan G-protein-coupled receptors: the next generation of drug targets?

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these `orphan'' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of `humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.     

Nonpeptide ligands that target peptide-activated GPCRs in inflammation

The focus of this review is on G protein-coupled receptors (GPCRs) for which nonpeptidic ligands are known and have been evaluated for the treatment of inflammatory conditions. GPCRs are the most prevalent class of cell surface proteins in pharmaceutical research today, and GPCR-targeting drugs account for one tenth of worldwide pharmaceutical sales. Of over 800 human GPCRs identified to date, several hundred are activated by peptides/proteins and just over 30 of these have been identified so far as potential therapeutic targets for the treatment of inflammatory diseases. This review highlights those GPCRs and over 60 structurally diverse nonpeptidic compounds that interact with them and display pro- or anti- inflammatory properties. Among these GPCR targets are the receptors for peptides like bradykinin, chemokines, complement anaphylatoxins, corticotropin releasing factor, endothelins, melanocortins, tachykinins, urocortins, as well as the protease activated receptors (PARs). Other peptide activated GPCRs implicated in inflammation, like those that bind angiotensin II, N-formyl peptides, galanin, neuropeptide Y, opioids and oxytocin, are only briefly discussed because there is either less direct association with inflammation or few/no nonpeptidic antiinflammatory ligands known. While it is still very early in the development of antiinflammatory drugs that target GPCRs, there is already a wealth of information supporting their important roles as cellular sentries in inflammatory diseases. New opportunities are emerging to evaluate antiinflammatory activities of potent and selective GPCR-binding ligands, including those being developed for other disease indications. In summary, GPCRs deserve a great deal more attention as potential therapeutic targets in inflammatory diseases.     

Tools for GPCR drug discovery

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.     

G protein-coupled receptors in drug discovery

G protein-coupled receptors (GPCRs) represent one of the most important drug discovery targets such that compounds targeted against GPCRs represent the single largest drug class currently on the market. With the revolutionary advances in human genome sciences and the identification of numerous orphan GPCRs, it is even more important to identify ligands for these orphan GPCRs so that their physiological and pathological roles can be delineated. To this end, major pharmaceutical industries are investing enormous amounts of time and money to achieve this object. This review is a bird's eye view on the various aspects of GPCRs in drug discovery.     

New perspectives regarding β(2) -adrenoceptor ligands in the treatment of asthma

In the last two decades several significant changes have been proposed in the receptor theory that describes how ligands can interact with G protein-coupled receptors (GPCRs). Here we briefly summarize the evolution of receptor theory and detail recent prominent advances. These include: (i) the existence of spontaneously active GPCRs that are capable of signalling even though they are unoccupied by any ligand; (ii) the discovery of ligands that can inactivate these spontaneously active receptors; (iii) the notion that a ligand may simultaneously activate more than one GPCR signalling pathway; and (iv) the notion that certain ligands may be able to preferentially direct receptor signalling to a specific pathway. Because the data supporting these receptor theory ideas are derived primarily from studies using artificial expression systems, the physiological relevance of these new paradigms remains in question. As a potential example of how these new perspectives in receptor theory relate to drug actions and clinical outcomes, we discuss their relevance to the recent controversy regarding the chronic use of β(2) -adrenoceptor agonists in the treatment of asthma.     

Gq protein-coupled receptors as targets for anesthetics

The mechanisms of action of anesthetics are unclear. Much attention has been focused on ion channels in the central nervous system as targets for anesthetics. During the last decade, major advances have been made in our understanding of the physiology and pharmacology of G-protein-coupled receptor (GPCR) signaling. Several lines of studies have shown that GPCRs are targets for anesthetics and that some anesthetics inhibit the functions of Gq-coupled receptors, including muscarinic acetylcholine (ACh) M(1), metabotropic type 5 glutamate, 5-hydroxytryptamine (5-HT) type 2A, and substance P receptors. Nearly 160 GPCRs have been identified, based on their gene sequence and ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs are targets for anesthetics, these oGPCRs represent a rich group of receptor targets for anesthetics. This article highlights the effects of anesthetics on Gq-coupled receptors, and discusses whether GPCRs other than Gq-coupled receptors are targets for anesthetics.     

An ultra-HTS process for the identification of small molecule modulators of orphan G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are the most successful target proteins for drug discovery research to date. More than 150 orphan GPCRs of potential therapeutic interest have been identified for which no activating ligands or biological functions are known. One of the greatest challenges in the pharmaceutical industry is to link these orphan GPCRs with human diseases. Highly automated parallel approaches that integrate ultra-high throughput and focused screening can be used to identify small molecule modulators of orphan GPCRs. These small molecules can then be employed as pharmacological tools to explore the function of orphan receptors in models of human disease. In this review, we describe methods that utilize powerful ultra-high-throughput screening technologies to identify surrogate ligands of orphan GPCRs.     

Insights into the pharmacological relevance of lysophospholipid receptors

The discovery of lysophospholipid (LP) 7-transmembrane, G protein-coupled receptors (GPCRs) that began in the 1990s, together with research into the functional roles of the major LPs known as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have opened new research avenues into their biological processes and mechanisms. Major examples of LP signalling effects include embryogenesis, nervous system development, vascular development, uterine implantation, immune cell trafficking, and inflammatory reactions. LP signalling also influences the pathophysiology of many diseases including cancer, autoimmune and inflammatory diseases, which indicate that LP receptors may be attractive targets for pharmacological therapies. A key example of such a therapeutic agent is the S1P receptor modulator FTY720, which upon phosphorylation and continued drug exposure, acts as an S1P receptor functional antagonist. This compound (also known as fingolimod or Gilenya) has recently been approved by the FDA for the treatment of relapsing forms of multiple sclerosis. Continued basic and translational research on LP signalling should provide novel insights into both basic biological mechanisms, as well as novel therapeutic approaches to combat a range of human diseases.     

Dopamine receptors – IUPHAR Review 13

The variety of physiological functions controlled by dopamine in the brain and periphery is mediated by the D₁, D₂, D₃, D₄ and D₅ dopamine GPCRs. Drugs acting on dopamine receptors are significant tools for the management of several neuropsychiatric disorders including schizophrenia, bipolar disorder, depression and Parkinson''s disease. Recent investigations of dopamine receptor signalling have shown that dopamine receptors, apart from their canonical action on cAMP-mediated signalling, can regulate a myriad of cellular responses to fine-tune the expression of dopamine-associated behaviours and functions. Such signalling mechanisms may involve alternate G protein coupling or non-G protein mechanisms involving ion channels, receptor tyrosine kinases or proteins such as β-arrestins that are classically involved in GPCR desensitization. Another level of complexity is the growing appreciation of the physiological roles played by dopamine receptor heteromers. Applications of new in vivo techniques have significantly furthered the understanding of the physiological functions played by dopamine receptors. Here we provide an update of the current knowledge regarding the complex biology, signalling, physiology and pharmacology of dopamine receptors.     

Techniques: promiscuous Galpha proteins in basic research and drug discovery

Assay technologies that measure the activation of heterotrimeric (alphabetagamma) G proteins by G-protein-coupled receptors (GPCRs) are well established within the pharmaceutical industry, either for pharmacological characterization or for the identification of natural or surrogate receptor ligands. Despite recent evidence indicating that GPCR-linked signalling events might not be mediated exclusively by G proteins, G-protein activation remains a common benchmark for assessing GPCR family members. Thus, assay systems that translate ligand-mediated modulation of GPCRs into G-protein-dependent intracellular responses still represent key components of both basic research and the drug discovery process. In this article, the current knowledge and recent progress of integrating Galpha subunits into assay systems for GPCR drug discovery will be reviewed. Emphasis is given to novel promiscuous and chimeric Galpha proteins. Because of their ability to interact with a wide range of GPCRs, such novel G proteins are likely to be incorporated rapidly into drug discovery programmes.     

Heterodimerization of g protein-coupled receptors: specificity and functional significance

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.     

GPCRs and cancer

G-protein-coupled receptors (GPCRs), which represent the largest gene family in the human genome, play a crucial role in multiple physiological functions as well as in tumor growth and metastasis. For instance, various molecules like hormones, lipids, peptides and neurotransmitters exert their biological effects by binding to these seven-transmembrane receptors coupled to heterotrimeric G-proteins, which are highly specialized transducers able to modulate diverse signaling pathways. Furthermore, numerous responses mediated by GPCRs are not dependent on a single biochemical route, but result from the integration of an intricate network of transduction cascades involved in many physiological activities and tumor development. This review highlights the emerging information on the various responses mediated by a selected choice of GPCRs and the molecular mechanisms by which these receptors exert a primary action in cancer progression. These findings provide a broad overview on the biological activity elicited by GPCRs in tumor cells and contribute to the identification of novel pharmacological approaches for cancer patients.     

Target validation of G-protein coupled receptors

G-protein coupled receptors (GPCRs) represent possibly the most important target class of proteins for drug discovery. Over 30% of clinically marketed drugs are active at this receptor family. These drugs exhibit their activity at <10% of all known GPCRs. A major challenge for the pharmaceutical industry is to associate the many novel GPCRs with disease to identify the drugs of the future. This process consists of a collection of experimental paradigms that together can be loosely labelled 'target validation'.     

A role for membrane potential in regulating GPCRs?

G-protein-coupled receptors (GPCRs) have ubiquitous roles in transducing extracellular signals into cellular responses. Therefore, the concept that members of this superfamily of surface proteins are directly modulated by changes in membrane voltage could have widespread consequences for cell signalling. Although several studies have indicated that GPCRs can be voltage dependent, particularly P2Y(1) receptors in the non-excitable megakaryocyte, the evidence has been mostly indirect. Recent work on muscarinic receptors has stimulated substantial interest in this field by reporting the first voltage-dependent charge movements for a GPCR. An underlying mechanism is proposed whereby a voltage-induced conformational change in the receptor alters its ability to couple to the G protein and thereby influences its affinity for an agonist. We discuss the strength of the evidence behind this hypothesis and include suggestions for future work. We also describe other examples in which direct voltage control of GPCRs can account for effects of membrane potential on downstream signals and highlight the possible physiological consequences of this phenomenon.     

Free fatty acid receptors and their physiological role in metabolic regulation

Free fatty acids (FFAs) are not only essential nutrient components, but they also function as signaling molecules in various physiological processes. In the progression of genomic analysis, many orphan G-protein coupled receptors (GPCRs) are found. Recently, GPCRs deorphanizing strategy successfully identified multiple receptors for FFAs. In these FFA receptors (FFARs), GPR40 (FFAR1) and GPR120 are activated by medium- to long- chain FFAs. GPR40 is expressed mainly in pancreatic β-cell and mediates insulin secretion, whereas GPR120 is expressed abundantly in the intestine and regulates the secretion of cholecystokinin (CCK) and glucagons-like peptide-1 (GLP-1), it promotes insulin secretion. Due to these biological activity, GPR40 and GPR120 are potential drug target for type 2 diabetes and selective ligands have been developed. In this review, we provide recent development in the field and discuss their physiological roles and their potential as drug targets.     

Conformational complexity of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human beta2 adrenergic receptor.     

N-Acyl amino acids and N-acyl neurotransmitter conjugates: neuromodulators and probes for new drug targets

The myriad functions of lipids as signalling molecules is one of the most interesting fields in contemporary pharmacology, with a host of compounds recognized as mediators of communication within and between cells. The N-acyl conjugates of amino acids and neurotransmitters (NAANs) have recently come to prominence because of their potential roles in the nervous system, vasculature and the immune system. NAAN are compounds such as glycine, GABA or dopamine conjugated with long chain fatty acids. More than 70 endogenous NAAN have been reported although their physiological role remains uncertain, with various NAAN interacting with a low affinity at G protein coupled receptors (GPCR) and ion channels. Regardless of their potential physiological function, NAAN are of great interest to pharmacologists because of their potential as flexible tools to probe new sites on GPCRs, transporters and ion channels. NAANs are amphipathic molecules, with a wide variety of potential fatty acid and headgroup moieties, a combination which provides a rich source of potential ligands engaging novel binding sites and mechanisms for modulation of membrane proteins such as GPCRs, ion channels and transporters. The unique actions of subsets of NAAN on voltage-gated calcium channels and glycine transporters indicate that the wide variety of NAAN may provide a readily exploitable resource for defining new pharmacological targets. Investigation of the physiological roles and pharmacological potential of these simple lipid conjugates is in its infancy, and we believe that there is much to be learnt from their careful study.