G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

G-protein-coupled receptor heteromers or how neurons can display differently flavoured patterns in response to the same neurotransmitter

It is becoming accepted that G-protein-coupled receptors (GPCRs) arrange in the neuronal membrane into homo- and hetero-oligomers and, therefore, these complexes mediate neurotransmission. New models are then needed to understand GPCR operation and predict the consequences of GPCR homo- or hetero-oligomerization. Although there is not any unifying theory addressing how hetero-oligomerization occurs, recent models have been devised to understand the thermodynamics of binding of neurotransmitters to GPCRs and the allosteric protomer-protomer interactions involved in neurotransmitter-mediated activation of GPCRs. Although a model to predict how signalling is produced via homo- or hetero-oligomerization is lacking, functional data show that receptor oligomers exist to produce a variety of effects in neurons in response to a single neurotransmitter.     

The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis,paralogon groups,and fingerprints

The superfamily of G-protein-coupled receptors (GPCRs) is very diverse in structure and function and its members are among the most pursued targets for drug development. We identified more than 800 human GPCR sequences and simultaneously analyzed 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. Our results show, with high bootstrap support, five main families, named glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin, forming the GRAFS classification system. The rhodopsin family is the largest and forms four main groups with 13 sub-branches. Positions of the GPCRs in chromosomal paralogons regions indicate the importance of tetraploidizations or local gene duplication events for their creation. We also searched for "fingerprint" motifs using Hidden Markov Models delineating the putative inter-relationship of the GRAFS families. We show several common structural features indicating that the human GPCRs in the GRAFS families share a common ancestor. This study represents the first overall map of the GPCRs in a single mammalian genome. Our novel approach of analyzing such large and diverse sequence sets may be useful for studies on GPCRs in other genomes and divergent protein families.     

Transmembrane signaling by GPCRs: Insight from rhodopsin and opsin structures

G-protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors, are the largest family of membrane proteins in the human genome. As versatile signaling molecules, they mediate cellular responses to extracellular signals. Diffusible ligands like hormones and neurotransmitters bind to GPCRs to modulate GPCR activity. An extraordinary and highly specialized GPCR is the photoreceptor rhodopsin which contains the chromophore retinal as its covalently bound ligand. For receptor activation the configuration of retinal is altered by photon absorption. To date, rhodopsin is the only GPCR for which crystal structures of inactive, active and ligand-free conformations are known. Although the photochemical activation is unique to rhodopsin, many mechanistic insights from this receptor can be generalized for GPCRs.     

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].     

Allostery at G protein-coupled receptor homo- and heteromers: uncharted pharmacological landscapes

For many years seven transmembrane domain G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomeric units. However, evidence both from native cells and heterologous expression systems has demonstrated that GPCRs can both traffic and signal within higher-order complexes. As for other protein-protein interactions, conformational changes in one polypeptide, including those resulting from binding of pharmacological ligands, have the capacity to alter the conformation and therefore the response of the interacting protein(s), a process known as allosterism. For GPCRs, allosterism across homo- or heteromers, whether dimers or higher-order oligomers, represents an additional topographical landscape that must now be considered pharmacologically. Such effects may offer the opportunity for novel therapeutic approaches. Allosterism at GPCR heteromers is particularly exciting in that it offers additional scope to provide receptor subtype selectivity and tissue specificity as well as fine-tuning of receptor signal strength. Herein, we introduce the concept of allosterism at both GPCR homomers and heteromers and discuss the various questions that must be addressed before significant advances can be made in drug discovery at these GPCR complexes.     

Formation of oligomers by G protein-coupled receptors

There is increasing evidence that G protein-coupled receptors (GPCRs) may form homo- or hetero-oligomers and that this may be important for their function. Evidence in favor of oligomerization comes from biochemical studies, studies of functional complementation and recent studies using energy transfer techniques, which provide direct evidence for receptor/receptor contacts. The oligomerization process may be regulated by ligands for some GPCRs or it may be constitutive in other cases. Homo-oligomerization may lead to cooperative behavior for the binding of ligands to some receptors. Hetero-oligomerization may generate new pharmacological properties for some receptor combinations as well as altering the signaling properties of the GPCR concerned. Additionally, the trafficking of GPCRs from the cell surface may be affected by oligomerization.     

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.     

Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.     

Heterodimerization and surface localization of G protein coupled receptors

G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: (1) how many functionally and pharmacologically distinct receptor subtypes exist in vivo? (2) Which GPCRs physically associate, and in what stochiometries? (3) What are the roles of individual subunits in binding ligand and activating responses? (4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease.     

GPCR-GIP networks: a first step in the discovery of new therapeutic drugs?

G protein-coupled receptors (GPCRs) are transmembrane molecules that, on interaction with G proteins upon ligand binding, can associate with a large variety of transmembrane or soluble proteins, termed 'GPCR-interacting proteins' (GIPs). Some special transmembrane GIPs are themselves GPCRs that form homo- or heterodimers, while other transmembrane GIPs are ionic channels, ionotropic receptors and single transmembrane proteins that control GPCR pharmacology and trafficking. Most soluble GIPs interact with the C-termini of GPCRs and often physically link GPCRs to large protein networks, called 'receptosomes', that include ionic channels, protein kinases, small G proteins, cytoskeletal proteins and adhesion molecules. Here, we review the nature and functions of some of these networks, such as the glutamate and serotonin receptosomes, and focus on the fine-tuning of GPCR signaling by GIPs. Finally, we discuss the possibilities for developing new therapeutic drugs capable of modulating GPCR signaling by disrupting or reinforcing specific GPCR-GIP interactions.     

G protein-coupled receptor drug discovery: implications from the crystal structure of rhodopsin

G protein-coupled receptors (GPCRs) are a functionally diverse group of membrane proteins that play a critical role in signal transduction. Because of the lack of a high-resolution structure, the heptahelical transmembrane bundle within the N-terminal extracellular and C-terminal intracellular region of these receptors has initially been modeled based on the high-resolution structure of bacterial retinal-binding protein, bacteriorhodopsin. However, the low-resolution structure of rhodopsin, a prototypical GPCR, revealed that there is a minor relationship between GPCRs and bacteriorhodopsins. The high-resolution crystal structure of the rhodopsin ground state and further refinements of the model provide the first structural information about the entire organization of the polypeptide chain and post-translational moieties. These studies provide a structural template for Family 1 GPCRs that has the potential to significantly improve structure-based approaches to GPCR drug discovery.     

Embracing emerging paradigms of G protein-coupled receptor agonism and signaling to address airway smooth muscle pathobiology in asthma

G protein-coupled receptors (GPCRs) regulate numerous airway cell functions, and signaling events transduced by GPCRs are important in both asthma pathogenesis and therapy. Indeed, most asthma therapies target GPCRs either directly or indirectly. Within recent years, our understating of how GPCRs signal and are regulated has changed significantly as new concepts have emerged and traditional ideas have evolved. In this review, we discuss current concepts regarding constitutive GPCR activity and receptor agonism, functional selectivity, compartmentalized signaling, and GPCR desensitization. We further discuss the relevance of these ideas to asthma and asthma therapy, while emphasizing their potential application to the GPCR signaling in airway smooth muscle that regulates airway patency and thus disease severity.     

Potential role of G protein-coupled receptor (GPCR) heterodimerization in neuropsychiatric disorders: A focus on depression

G protein-coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and are the target of approximately half of all therapeutic drugs. For many years, GPCRs were thought to exist and function as monomeric units. However, during the past two decades, substantial biochemical, structural and functional evidence have indicated that GPCRs can associate and form heteromers that exhibit functional properties distinct from the corresponding monomers. The understanding of the unique pharmacological and functional properties of such heteromers is a major challenge for neuroscience, particularly given the abundant evidence suggesting that GPCR heteromers may play a crucial role in neuropsychiatric disorders. Herein, we present current data on the role of GPCR heterodimerization in neuropsychiatric disorders, with a focus on its potential implications in depression. The presented examples of pairs of receptors, with their specific pharmacological and functional properties, are likely to lead to novel effective strategies in antidepressant drug development. The currently available techniques for studying GPCR heterodimerization, both in vitro as well as in situ in native tissue, are also described.     

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.     

Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors

The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.     

New G-protein-coupled receptor crystal structures: insights and limitations

G-protein-coupled receptors (GPCRs) constitute a large family of structurally similar proteins that respond to a chemically diverse array of physiological and environmental stimulants. Until recently, high-resolution structural information was limited to rhodopsin, a naturally abundant GPCR that is highly specialized for the detection of light. Non-rhodopsin GPCRs for diffusible hormones and neurotransmitters have proven more resistant to crystallography approaches, possibly because of their inherent structural flexibility and the need for recombinant expression. Recently, crystal structures of the human beta(2) adrenoceptor have been obtained using two different approaches to stabilize receptor protein and increase polar surface area. These structures, together with the existing structures of rhodopsin, represent an important first step in understanding how GPCRs work at a molecular level. Much more high-resolution information is needed for this important family of membrane proteins, however: for example, the structures of GPCRs from different families, structures bound to ligands having different efficacies, and structures of GPCRs in complex with G proteins and other signaling molecules. Methods to characterize the dynamic aspects of the GPCR architecture at high resolution will also be important.     

Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR.     

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.