Dimerization of chemokine receptors in living cells: key to receptor function and novel targets for therapy

Chemokine receptors control and mediate a diverse array of physiological and pathogenic processes. Many seven transmembrane (TM) G-protein-coupled receptors (GPCRs), including chemokine receptors, exist as homo- or heterodimers. Growing evidence indicates that the dimeric form is the basic functional structure of these receptors. Hetero-dimerization may allow for enhanced or specific functions of receptors and may be essential for receptor activity. Thus, dimers may provide new targets for chemokine receptor-based therapies. Synthetic peptides of TM regions of chemokine receptors may interfere with homologous interactions and inhibit functional activity of the receptors. Therefore, TM peptides and possibly compounds that target dimers and/or signaling of chemokine receptors may have therapeutic applications.     

Allosteric modulation of heterodimeric G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are, and will probably remain, the most tractable class of targets for the development of small-molecule therapeutic medicines. Currently, all approved GPCR-directed medicines are agonists or antagonists at orthosteric binding sites - except for the calcimimetic cinacalcet, which is a positive allosteric modulator of Ca(2+)-sensing receptors, and maraviroc, an allosteric inhibitor of CC-chemokine receptor (CCR) 5. It is now widely accepted that GPCRs exist and might function as dimers, and there is growing evidence for the physiological presence and relevance of GPCR heterodimers. Molecules that can regulate a GPCR within a heterodimer, through allosteric effects between the two protomers of the dimer or between a protomer or protomers and the associated G protein, offer the potential to function in a highly selective and tissue-specific way. Despite the conceptual attraction of such allosteric regulators of GPCR heterodimers as drugs, they cannot be identified by screening approaches that routinely use a 'one GPCR target at a time' strategy. In our opinion, this will require the development of new approaches for screening and a return to the use of physiologically relevant cell systems at an early stage in compound identification.     

Physiological relevance of GPCR oligomerization and its impact on drug discovery

The potentially large functional and physiological diversity of G-protein coupled receptor (GPCR) dimers has generated a great deal of excitement about the opportunity that dimerization provides for enabling novel drug discovery. The discovery of physiologically relevant GPCR dimers suggests that new drug targets for diseases such as schizophrenia and pre-eclampsia can be developed by targeting dimers. Most of the previous work on GPCR dimers made use of the overexpression of differentially tagged GPCRs in heterologous cell systems. Current emphasis on the development of physiologically relevant cell systems that endogenously express the appropriate combination of GPCR dimers and accessory proteins is leading to dramatic increases in our understanding of GPCR dimers. These and other new tools such as GPCR-specific antibodies will be required to develop GPCR dimer specific drugs. Given that ligands are available for only a small percentage of the large number of potentially druggable GPCRs, the use of GPCR dimers might provide the necessary targets to increase the breadth and depth of receptors available for therapeutic interventions.     

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.     

G-protein-coupled receptor heteromers: function and ligand pharmacology

Almost all existing models for G-protein-coupled receptors (GPCRs) are based on the occurrence of monomers. Recent studies show that many GPCRs are dimers. Therefore for some receptors dimers and not monomers are the main species interacting with hormones/neurotransmitters/drugs. There are reasons for equivocal interpretations of the data fitting to receptor dimers assuming they are monomers. Fitting data using a dimer-based model gives not only the equilibrium dissociation constants for high and low affinity binding to receptor dimers but also a 'cooperativity index' that reflects the molecular communication between monomers within the dimer. The dimer cooperativity index (D(C)) is a valuable tool that enables to interpret and quantify, for instance, the effect of allosteric regulators. For different receptors heteromerization confers a specific functional property for the receptor heteromer that can be considered as a 'dimer fingerprint'. The occurrence of heteromers with different pharmacological and signalling properties opens a complete new field to search for novel drug targets useful to combat a variety of diseases and potentially with fewer side effects. Antagonists, which are quite common marketed drugs targeting GPCRs, display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs.     

G蛋白偶联受体二聚化在药物开发中所发挥的特异性作用(英文)

G蛋白偶联受体(GPCRs)是与G蛋白相偶联的七次跨膜受体,其成员有上千种,是重要的药物靶点之一。目前,GPCRs相关药物占市场上药物的40%-50%。在过去的十年中,对GPCRs主要以单体的形式存在着的这一假说做出了重新评估,大量事实证明GPCRs也能以同源或异源二聚体,甚至是高阶寡聚体的形式存在,比较热门的领域是GPCRs二聚化。最近研究表明同源或异源二聚化有不同于单体的特异功能特征,包括配体识别、信号转导、运输等。同时,在较少副作用治疗疾病的新药开发上,具有不同病理和信号转导途径的二聚体的出现开辟了新的领域。本综述主要介绍二聚体的特异结构及其特异的信号转导途径,从而有助于在GPCRs药物开发中取得丰硕的成果。     

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 ligand-dendrimer (GLiDe) conjugates: future smart drugs?

Unlike nanocarriers that are intended to release their drug cargo at the site of action, biocompatibile polyamidoamine (PAMAM) conjugates are designed to act at cell surface G protein-coupled receptors (GPCRs) without drug release. These multivalent GPCR ligand-dendrimer (GLiDe) conjugates display qualitatively different pharmacological properties in comparison with monomeric drugs. They might be useful as novel tools to study GPCR homodimers and heterodimers as well as higher aggregates. The structure of the conjugate determines the profile of biological activity, receptor selectivity, and physical properties such as water solubility. Prosthetic groups for characterization and imaging of receptors can be introduced without loss of affinity. The feasibility of targeting multiple adenosine and P2Y receptors for synergistic effects has been shown. Testing in vivo will be needed to explore the effects on pharmacokinetics and tissue targeting.     

The structure and dynamics of GPCR oligomers: a new focus in models of cell-signaling mechanisms and drug design

G protein-coupled receptors (GPCRs) represent approximately half of the potential pharmaceutical targets for current drugs, and thus the way in which these receptors assemble into dimeric/oligomeric structures is of vital interest in practical as well as conceptual aspects of current drug discovery efforts. The significance of such structures is based on the recent realization that ligand-dependent signaling by GPCRs is not necessarily transduced to the G protein by receptor monomers, but possibly by GPCR dimers or even oligomers that function as dynamic macromolecular assemblies. In addition, recent evidence that GPCR hetero-oligomerization can produce signaling units with unexpected combinations of pharmacological properties suggests entirely new methods for developing successful drugs. The dynamic mechanisms of these signaling assemblies remain to be elucidated. The development of increasingly accurate dynamic molecular models of GPCR dimers is expected to produce a more complete structural context for understanding the molecular mechanisms of GPCR function, and to aid in drug discovery.     

Computational and Experimental Analysis of the Transmembrane Domain 4/5 Dimerization Interface of the Serotonin 5-HT1A Receptor

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT(1A) receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-F?rster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp175(4.64), Tyr198(5.41), Arg151(4.40), and Arg152(4.41)) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175(4.64) in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT(1A) receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.     

Probing the existence of G protein-coupled receptor dimers by positive and negative ligand-dependent cooperative binding

An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerization could be a possible cross-talk between the protomers, which in turn could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation, and competition binding experiments were performed on vasopressin and oxytocin receptors expressed in Chinese hamster ovary or COS-7 cells. Linear, concave, and convex Scatchard plots were then obtained, depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors, suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results, which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.     

Regulation of G protein-coupled receptor function by its binding proteins

G protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A(2) receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPalpha and TPbeta. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28gamma and proteasome subunit alpha7, were identified as direct interacting proteins for TPbeta. TPbeta has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPalpha and TPbeta formed heterodimers, and the signal transduction through TPalpha was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.     

G-protein-coupled receptor heterodimers: pharmacology, function and relevance to drug discovery

The growing recognition that members of the rhodopsin-like family A G-protein-coupled receptors (GPCRs) exist and function as dimers or higher-order oligomers, and that GPCR hetero-dimers and -oligomers are present in physiological tissues, offers novel opportunities for drug discovery. Differential pharmacology, function and regulation of GPCR hetero-dimers and -oligomers suggest means to selectively target GPCRs in different tissues and hint that the mechanism of function of several pharmacological agents might be different in vivo than anticipated from simple ligand-screening programmes that rely on heterologous expression of a single GPCR.     

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.     

Computational methods in drug design: Modeling G protein-coupled receptor monomers, dimers, and oligomers

G protein-coupled receptors (GPCRs) are membrane proteins that serve as very important links through which cellular signal transduction mechanisms are activated. Many vital physiological events such as sensory perception, immune defense, cell communication, chemotaxis, and neurotransmission are mediated by GPCRs. Not surprisingly, GPCRs are major targets for drug development today. Most modeling studies in the GPCR field have focused upon the creation of a model of a single GPCR (ie, a GPCR monomer) based upon the crystal structure of the Class A GPCR, rhodopsin. However, the emerging concept of GPCR dimerization has challenged our notions of the monomeric GPCR as functional unit. Recent work has shown not only that many GPCRs exist as homo- and heterodimers but also that GPCR oligomeric assembly may have important functional roles. This review focuses first on methodology for the creation of monomeric GPCR models. Special emphasis is given to the identification of localized regions where the structure of a GPCR may diverge from that of bovine rhodopsin. The review then focuses on GPCR dimers and oligomers and the bioinformatics methods available for identifying homo- and heterodimer interfaces.     

Targeting opioid receptor heterodimers: Strategies for screening and drug development

G-protein-coupled receptors are a major target for the development of new marketable drugs. A growing number of studies have shown that these receptors could bind to their ligands, signal, and be internalized as dimers. Most of the evidence comes from in vitro studies, but recent studies using animal models support an important role for dimerization in vivo and in human pathologies. It is therefore becoming highly relevant to include dimerization in screening campaigns: the increased complexity reached by the ability to target 2 receptors should lead to the identification of more specific hits that could be developed into drugs with fewer side effects. In this review, we have summarized results from a series of studies characterizing the properties of G-protein-coupled receptor dimers using both in vitro and in vivo systems. Since opioid receptors exist as dimers and heterodimerization modulates their pharmacology, we have used them as a model system to develop strategies for the identification of compounds that will specifically bind and activate opioid receptor heterodimers: such compounds could represent the next generation of pain relievers with decreased side effects, including reduced drug abuse liability.     

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.     

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.     

Emerging roles for orphan G-protein-coupled receptors in the cardiovascular system

Despite current drug therapies, including those that target enzymes, channels and known G-protein-coupled receptors (GPCRs), cardiovascular disease remains the major cause of ill health, which suggests that other transmitter systems might be involved in this disease. In humans, approximately 175 genes have been predicted to encode 'orphan' GPCRs, where the endogenous ligand is not yet known. As a result of intensive screening using 'reverse pharmacology', an increasing number of orphan receptors are being paired with their cognate ligands, many of which are peptides. The existence of some of these peptides such as urotensin-II and relaxin had been known for some time but others, including ghrelin and apelin, represent novel sequences. The pharmacological characterization of these emerging peptide-receptor systems is a tantalising area of cardiovascular research, with the prospect of identifying new therapeutic targets.     

Asymmetry of GPCR oligomers supports their functional relevance

G protein-coupled receptors (GPCRs) can exist as dimers or as larger oligomeric clusters that enable intercommunication between different receptor protomers within the same complex. This phenomenon is observed at three distinct levels: (i) at the level of ligand binding where the activation of one protomer can allosterically inhibit or facilitate ligand binding to the second protomer; (ii) at the level of ligand-induced conformational switches, which occur between transmembrane domains of the two protomers; and (iii) within GPCR-associated protein complexes, either directly at the level of GPCR-interacting proteins or at further downstream levels of the complex. Intercommunication at these different levels introduces asymmetry within GPCR dimers wherein each protomer fulfills its specific task. In this review, we discuss how the asymmetric behavior of GPCRs highlights the advantage of oligomeric receptor organization and supports the functional relevance of GPCR dimerization.