Localization of G-protein-coupled receptors in health and disease

G-protein-coupled receptors (GPCRs) represent a superfamily of proteins, characterized by seven transmembrane alpha-helices, that signal through interactions with a family of heterotrimeric GTP-binding proteins, referred to as G proteins. The broad range of physiological functions associated with GPCRs indicates that a better understanding of these receptors and their regulation can provide a solid foundation for novel pharmacological interventions in a variety of disease states.     

Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.     

Historical review: Negative efficacy and the constitutive activity of G-protein-coupled receptors

The idea that a receptor can produce signalling without agonist intervention and that several antagonists can be 'active' in repressing such spontaneous activity is contained in the concept of ligand-induced conformational changes. Yet, this idea was neglected by pharmacologists for many years. In this article, we review the events that brought inverse agonism and constitutive activity to general attention and made this phenomenon a topic of current research. We also suggest a classification of antagonists based on the cooperativity that links their primary site of interaction with other functional domains of the receptor.     

Odorant receptors: a plethora of G-protein-coupled receptors.

Odorant receptors (ORs) comprise the largest family of G-protein-coupled receptors (GPCRs). They are located in the nasal epithelium, at the ciliated surface of olfactory sensory neurones, where the initial steps of the olfactory transduction cascade occur. ORs are encoded by a large and diverse multi-gene family, which has been characterized in cyclostomes, teleosts, amphibia, birds and mammals, as well as in Drosophila and Caenorhabditis elegans. Here, the range of diversity in OR and chemoreceptor structure is examined, noting that their functions are fundamentally similar to those of many neurotransmitter or neurohormone receptors. It is argued that ORs have emerged directly from other GPCRs independently in many species. According to this view, there is no structural prerequisite for OR identity and any GPCR has the potential to be or become an OR at a given point in evolution.     

G-protein-coupled receptors: past, present and future

The G-protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Drugs active at these receptors have therapeutic actions across a wide range of human diseases ranging from allergic rhinitis to pain, hypertension and schizophrenia. This review provides a brief historical overview of the properties and signalling characteristics of this important family of receptors.     

Dimerization of G-protein-coupled receptors.

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.     

Postgenomic characterization of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). In this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.     

Allosteric modulators of G-protein-coupled receptors

Allosteric modulators of G-protein-coupled receptors (GPCRs) interact with binding sites on the receptor that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Allosteric modulators offer several advantages over standard orthosteric drugs, including the potential for greater receptor subtype selectivity. To date, the current paucity of clinically available allosteric drugs reflects the bias of traditional radioligand binding assays towards the detection of orthosteric effects. However, the advent of new cell-based high-throughput functional assays has led to an increased detection of allosteric GPCR ligands. The current challenge for modulator-based GPCR drug discovery is the optimization of both binding and functional assays to better detect and validate allosteric ligands.     

Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective

Over the last decade, the ability to detect agonist-independent signal transduction by G protein-coupled receptors has in turn resulted in the detection and study of ligands able to block this activity. Such ligands are generically described as inverse agonists. Considerable attention has recently been devoted to the presence and roles of endogenous antagonist/inverse agonists and the concept that inverse agonists may have specific therapeutic benefits compared with neutral antagonists.     

Allosteric modulation of G-protein-coupled receptors

Allosteric modulation of G-protein-coupled receptors may provide an alternative approach for selective receptor interactions. The article is an overview of allosteric modulators enhancing or diminishing the effects of (endogenous) agonists or antagonists on a variety of G-protein-coupled receptors such as muscarinic, α-adrenergic, serotoninergic, dopaminergic, adenosine, metabotropic glutamate and Ca²⁺ receptors. Efficacious allosteric modulators have been published for the serotonin receptor (5-HT moduline; oleamide), the dopamine receptor (cyclic analogues of the tripeptide prolyl-leucyl-glycinamide), the metabotropic glutamate receptor (CPCCOET (mglu1); MPEP (mglu5)) and the Ca²⁺ receptor (NPS R-568; NPS 2143). Leads are available for the muscarinic (Ach) receptor (K5720) and the α_2A-adrenoceptor (agmatine). SCH-202676 and amiloride analogues are nonselective allosteric modifiers interacting with several different receptors. They may not be suitable as leads for future drugs but such compounds may be used for screening purposes.     

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.     

Linking Chinese medicine and G-protein-coupled receptors

Following the purification of the immunosuppressant ISP-1 from a Chinese medicine, Japanese scientists have developed a more potent immune modulator, FTY720, that induces T-cell homing. FTY720, a promising immunosuppressant for use in patients with tissue transplants and autoimmune diseases, is currently in clinical trials. Two recent studies have elucidated that the mechanism of action of FTY720 is via a subset of G-protein-coupled receptors for the lysophospholipid mediator sphingosine-1-phosphate.     

G-protein-coupled receptors and signaling networks: emerging paradigms.

G-protein-coupled receptors (GPCRs) constitute the largest family of cell-surface molecules involved in signal transmission. These receptors play key physiological roles and their dysfunction results in several diseases. Recently, it has been shown that many of the cellular responses mediated by GPCRs do not involve the sole stimulation of conventional second-messenger-generating systems, but instead result from the functional integration of an intricate network of intracellular signaling pathways. Effectors for GPCRs that are independent of G proteins have now also been identified, thus changing the conventional view of the GPCR-heterotrimeric-G-protein-associated effector. The emerging information is expected to help elucidate the most basic mechanism by which these receptors exert their numerous physiological roles, in addition to determining why the perturbation of their function results in many pathological conditions.     

Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking

Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the 'novel pharmacology' and functional implications resulting from heterodimerization will be discussed.     

Identification of G-protein-coupled receptors involved in inflammatory disease by genetic association studies

G-protein-coupled receptors are not only highly tractable drug targets but also attractive candidates for genetic association studies because they are more polymorphic than most other classes of gene and these polymorphisms frequently lead to functional changes in the levels of expression or biological activity that can predispose to common diseases. A large-scale study to identify functional variants in G-protein-coupled receptors associated with inflammatory diseases has highlighted a spectrum of novel biological insights that range from identifying the involvement of orphan receptors in certain diseases through to highlighting new therapeutic indications for existing drugs.     

Trafficking of delta-opioid receptors and other G-protein-coupled receptors: implications for pain and analgesia

A cell can regulate how it interacts with its external environment by controlling the number of plasma membrane receptors that are accessible for ligand stimulation. G-protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptors and have a significant role in physiological and pathological processes. Much research effort is now focused on understanding how GPCRs are delivered to the cell surface to enhance the number of 'bioavailable' receptors accessible for activation. Knowing how such processes are triggered or modified following induction of various pathological states will inevitably identify new therapeutic strategies for treating various diseases, including chronic pain. Here, we highlight recent advances in this field, and provide examples of the importance of such trafficking events in pain.