Structural mechanism of GPCR-arrestin interaction: recent breakthroughs

G protein-coupled receptors (GPCRs) are a major membrane receptor family with important physiological and pathological functions. In the classical signaling pathway, ligand-activated GPCRs couple to G proteins, thereby inducing G protein-dependent signaling pathways and phosphorylation by G protein-coupled receptor kinases (GRKs). This leads to an interaction with arrestins, which results in GPCR desensitization. Recently, non-classical GPCR signaling pathways, mediated by GPCR-bound arrestins, have been identified. Consequently, arrestins play important roles in GPCR signaling not only with respect to desensitization but also in relation to G protein-independent signal transduction. These findings have led to efforts to develop functionally biased (i.e. signal transduction biased) GPCR-targeting drugs. One of these efforts is aimed at understanding the structural mechanism of functionally biased GPCR signaling, which includes understanding the G protein-selectivity or arrestin-selectivity of GPCRs. This goal has not yet been achieved; however, great progress has been made during the last 3 years toward understanding the structural mechanism of GPCR-mediated arrestin activation. This review will discuss the recent breakthroughs in the conformational understanding of GPCR-arrestin interaction.     

Structure-based drug screening for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past 4 years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.     

Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery

During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein-coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in-vivo manipulation of levels of GPCRs using transgenic and gene knock-out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology. Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.     

Structural insights into RAMP modification of secretin family G protein-coupled receptors: implications for drug development

Secretin family G protein-coupled receptors (GPCRs) are important therapeutic targets for migraine, diabetes, bone disorders, inflammatory disorders and cardiovascular disease. They possess a large N-terminal extracellular domain (ECD) known to be the primary ligand-binding determinant. Structural determination of several secretin family GPCR ECDs in complex with peptide ligands has been achieved recently, providing insight into the molecular determinants of hormone binding. Some secretin family GPCRs associate with receptor activity-modifying proteins (RAMPs), resulting in changes to receptor pharmacology. Recently, the first crystal structure of a RAMP ECD in complex with a secretin family GPCR was solved, revealing the elegant mechanism governing receptor selectivity of small molecule antagonists of the calcitonin gene-related peptide (CGRP) receptor. Here we review the structural basis of ligand binding to secretin family GPCRs, concentrating on recent progress made on the structural basis of RAMP-modified GPCR pharmacology and its implications for rational drug design.     

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.     

Conformational changes in G-protein-coupled receptors-the quest for functionally selective conformations is open

The G-protein-coupled receptors (GPCRs) represent one the largest families of drug targets. Upon agonist binding a receptor undergoes conformational rearrangements that lead to a novel protein conformation which in turn can interact with effector proteins. During the last decade significant progress has been made to prove that different conformational changes occur. Today it is mostly accepted that individual ligands can induce different receptor conformations. However, the nature or molecular identity of the different conformations is still ill-known. Knowledge of the potential functionally selective conformations will help to develop drugs that select specific conformations of a given GPCR which couple to specific signalling pathways and may, ultimately, lead to reduced side effects. In this review we will summarize recent progress in biophysical approaches that have led to the current understanding of conformational changes that occur during GPCR activation.     

G-protein-coupled receptor oligomerization and its potential for drug discovery

G-protein-coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. Virtually all therapeutics that are directed towards GPCRs have been designed using assays that presume that these receptors are monomeric. The recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. However, this important aspect of GPCR biology remains largely unincorporated into schemes to search for new therapeutics. This review provides a synopsis of the current thinking surrounding GPCR homo-oligomerization and hetero-oligomerization and shows how new models point towards unexplored avenues in the development of new therapies.     

Recent advances in molecular pharmacology of the histamine systems: roles of C-terminal tails of histamine receptors

G-protein-coupled receptors (GPCR) represent a large and diverse superfamily of integral membrane proteins to which histamine receptors belong. Increasing numbers of proteins have been identified to interact with the C-termini of GPCRs. These interactions are implicated in targeting, trafficking, and fine-tuning of signaling of GPCRs. Although the C-terminus of the histamine H2 receptor has been suggested to play in agonist-induced internalization of the receptor, roles of the C-termini of the other three histamine receptors are not known. To date, there is no protein identified to interact with the C-termini of histamine receptors.     

Orphan G-protein-coupled receptors : strategies for identifying ligands and potential for use in eating disorders

G-protein-coupled receptors (GPCRs) are key regulators of intercellular interactions, participating in almost every physiological response. They exert their effects by being activated by a variety of endogenous ligands. Traditionally, these ligands were identified first, providing tools to characterise the receptors. However, since the late 1980s, homology screening approaches have allowed the GPCRs to be found first, and in turn used as orphan targets to identify their ligands. Over the last decade this method has led to the identification of 12 novel neuropeptide families. Interestingly, four of these deorphanised GPCR systems, melanin-concentrating hormone, ghrelin, orexin and neuropeptide B/neuropeptide W, have been found to play a role in the control of energy balance. This article reviews the role of these GPCR systems in the control of food intake and energy expenditure, and discusses their potential use in therapies directed at eating disorders. As obesity has reached epidemic proportions across the developed world, pharmacotherapy has focused on this condition. However, difficulties in weight control also characterise disorders of binge eating such as bulimia and binge-eating disorder. Consequently, hypophagic treatments may be of potential benefit in normal, overweight or obese individuals displaying aberrant (out of control) eating behaviour.     

Receptor tyrosine kinase-G-protein-coupled receptor signalling platforms: out of the shadow?

Receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs) can form platforms in which protein signalling components specific for each receptor are shared (owing to close proximity) to produce an integrated response upon engagement of ligands. RTK-GPCR signalling platforms respond to growth factors and GPCR agonists to increase gain over and above that which is normally produced by separate receptors. They can also function to change the spatial context of signalling in response to growth factor activation. The function of RTK-GPCR signalling platforms can be modulated with conformational-specific inhibitors that stabilise defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses. In this paper, we provide an opinion of the biology and unusual pharmacology of RTK-GPCR signalling platforms and make comparisons with a more traditional model of crosstalk between RTKs and GPCRs.     

Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation

The idea that G-protein-coupled receptors (GPCRs) can function as dimers is now generally accepted. Although an increasing amount of data suggests that dimers represent the basic signaling unit for most, if not all, members of this receptor family, GPCR dimerization might also be necessary to pass quality-control checkpoints of the biosynthetic pathway of GPCRs. To date, this hypothesis has been demonstrated unambiguously only for a small number of receptors that must form heterodimers to be exported properly to the plasma membrane (referred to as obligatory heterodimers). However, increasing evidence suggests that homodimerization might have a similar role in the receptor maturation process for many GPCRs.     

A closer look into G protein coupled receptor activation: X-ray crystallography and long-scale molecular dynamics simulations

G protein coupled receptors (GPCRs) are a large eukaryotic protein family of transmembrane receptors that react to a signal coming from the extracellular environment to generate an intracellular response through the activation of a signal transduction pathway mediated by a heterotrimeric G protein. Their diversity, dictated by the multiplicity of stimuli to which they respond and by the variety of intracellular signalling pathways they activate, make them one of the most prominent families of validated pharmacological targets in biomedicine. In recent years, major breakthroughs in structure determination of GPCRs have given new stimuli to the exploration of the biology of these proteins, providing a structural basis to understand the molecular origin of GPCR mechanisms of action. Based on the information coming from these structural studies, a number of recent in silico investigations used molecular dynamics (MD) simulations to contribute to our knowledge of GPCRs. In this review, we will focus on investigations that, taking advantage of the tremendous progress in both hardware and software, made testable hypotheses that have been validated by subsequent structural studies. These stateof- the-art molecular simulations highlight the potential of microsecond MD simulations as a valuable tool in GPCR structural biology and biophysics.     

Constitutive desensitization: a new paradigm for g protein-coupled receptor regulation

GPCRs are a large family of cell-surface proteins that regulate many important biochemical pathways and physiological responses. The isolation and characterization of GPCRs represent one of the more remarkable success stories that occurred during the revolution in biology of the last quarter century. Of the many discoveries that originated in the laboratory of Robert Lefkowitz at Duke University concerning GPCR regulation, none is more fundamental than the elucidation of the families of GRKs and arrestin proteins that terminate GPCR signaling. In this essay, we will discuss how advances in microscopy and biology have made the visualization of GPCR, GRK, and arrestin activity possible in single cells. Additionally, we will discuss how imaging studies using arrestins and a naturally occurring mutant of the vasopressin receptor led to the recognition of a novel phenotypic receptor behavior, in which the receptor desensitizes in the absence of agonist. We have termed this process constitutive desensitization, and this unexpected receptor property suggests that it may be possible to develop novel classes of signal-inhibiting drugs distinct from conventional antagonists.     

X-ray structure breakthroughs in the GPCR transmembrane region

G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25-50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993-6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021-3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven α-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734-40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1-12].     

Rich tapestry of G protein-coupled receptor signaling and regulatory mechanisms

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins and the most common therapeutic targets. In the last 2 decades, impressive progress in the understanding of GPCR function has been achieved, driven largely by the idea of similarity of the molecular mechanisms underlying their signaling and regulation. However, recent comprehensive studies of signaling and trafficking of several GPCR subtypes, including endogenous M3 muscarinic and H1 histamine receptor and expressed cysteinyl leukotriene type 1 receptor in human embryonic kidney 293 cells, clearly demonstrate that each receptor is regulated by a unique set of molecular mechanisms involving different players. These data indicate that the "gold mine" of similarities is nearly exhausted and that extrapolation from one receptor to another is as likely to be misleading as illuminating. Further progress in the field requires careful analysis of the regulation of individual GPCR subtypes in defined cellular context. In this issue of Molecular Pharmacology, Luo et al. (p. 338) describe a complex pattern of the regulation of M3 muscarinic receptor signaling.     

Therapeutic implications of endothelin and thrombin G‐protein‐coupled receptor transactivation of tyrosine and serine/threonine kinase cell surface receptors

Objectives This review discusses the latest developments in G protein coupled receptor (GPCR) signalling related to the transactivation of cell surface protein kinase receptors and the therapeutic implications. Key findings Multiple GPCRs have been known to transactivate protein tyrosine kinase receptors for almost two decades. More recently it has been discovered that GPCRs can also transactivate protein serine/threonine kinase receptors such as that for transforming growth factor (TGF)‐β. Using the model of proteoglycan synthesis and glycosaminoglycan elongation in human vascular smooth muscle cells which is a component of an in vitro model of atherosclerosis, the dual tyrosine and serine/threonine kinase receptor transactivation pathways appear to account for all of the response to the agonists, endothelin and thrombin. Summary The broadening of the paradigm of GPCR receptor transactivation explains the broad range of activities of these receptors and also the efficacy of GPCR antagonists in cardiovascular therapeutics. Deciphering the mechanisms of transactivation with the aim of identifying a common therapeutic target remains the next challenge.     

Conformational changes involved in G-protein-coupled-receptor activation

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M(3) muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.     

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.     

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.     

Modeling the 3D structure of GPCRs: advances and application to drug discovery

G protein-coupled receptors (GPCRs) are membrane-embedded proteins responsible for signal transduction; these receptors are, therefore, among the most important pharmaceutical drug targets. In the absence of X-ray structures, there have been numerous attempts to model the three-dimensional (3D) structure of GPCRs. In this review, the current status of GPCR modeling is evaluated, highlighting recent progress made in rhodopsin-based homology modeling and de novo modeling technology. Assessment of recent rhodopsin-based homology modeling studies indicates that, despite significant progress, these models do not yield hit rates that are sufficiently high for in silico screening (10 to 40% when screening for known binders). In contrast, the PREDICT modeling algorithm, which is independent of the rhodopsin structure, has now been fully validated in the context of drug discovery. PREDICT models are successfully used for drug discovery, yielding excellent hit rates (85 to 100% when screening for known binders), leading to the discovery of nanomolar-range new chemical entities for a variety of GPCR targets. Thus, 3D models of GPCRs should now allow the use of productive structure-based approaches for drug discovery.