G Protein-coupled Receptors Heterodimerization Research Articles

Heterodimerization of Hypothalamic G-Protein-Coupled Receptors Involved in Weight Regulation

Background: Melanocortin 3 and 4 receptors (MC3R and MC4R) are known to play an essential role in hypothalamic weight regulation. In addition to these two G-protein-coupled receptors (GPCRs), a huge number of other GPCRs are expressed in hypothalamic regions, and some of them are involved in weight regulation. So far, homodimerization was shown for a few of these receptors. Heterodimerization of unrelated receptors may have profound functional consequence but heterodimerization of GPCRs involved in weight regulation was not reported yet. Methods: A selective number of hypothalamically expressed GPCRs were cloned into a eukaryotic expression vector. Cell surface expression was demonstrated by an ELISA approach. Subcellular distribution was investigated by confocal laser microscopy. A sandwich ELISA and fluorescence resonance energy transfer (FRET) were used to determine protein-protein interaction. Results: Via sandwich ELISA and FRET approach we could demonstrate a robust interaction of the MC4R with GPR7, both of which are expressed in the hypothalamic nucleus paraventricularis. Moreover, we determined a strong interaction of MC3R with the growth hormone secretagogue receptor expressed in the nucleus arcuatus. Conclusion: Identification GPCR heterodimerization adds to the understanding of the complexity of weight regulation and may provide important information to develop therapeutic strategies to treat obesity.

Opioid and chemokine receptor heterodimers: arranged marriages or dangerous liaisons?

Heterodimerization of GPCRs (G-protein-coupled receptors) has begun to be appreciated as a rich new vein for drug discovery. The possibility of developing modulators for different GPCRs which work at allosteric sites other than the ligand-binding site is not new, but the notion of using a dimeric receptor partner as the target for this intervention has not yet percolated into the broader industrial community. In the present article, I discuss this notion in the context of the heterodimeric delta-opioid receptor-CXCR2 chemokine receptor dimer identified by Parenty et al., in this issue of the Biochemical Journal.

Constitutive Activity of the Cannabinoid CB1 Receptor Regulates the Function of Co-expressed Mu Opioid Receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

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.

CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene–dependent mitogenic responses of mast cells

AbstractCysteinyl leukotrienes (cys-LTs) induce inflammation through 2 G protein–coupled receptors (GPCRs), CysLT1 and CysLT2, which are coexpressed by most myeloid cells. Cys-LTs induce proliferation of mast cells (MCs), transactivate c-Kit, and phosphorylate extracellular signal-regulated kinase (ERK). Although MCs express CysLT2, their responses to cys-LTs are blocked by antagonists of CysLT1. We demonstrate that CysLT2 interacts with CysLT1, and that knockdown of CysLT2 increases CysLT1 surface expression and CysLT1-dependent proliferation of cord blood–derived human MCs (hMCs). Cys-LT–mediated responses were absent in MCs from mice lacking CysLT1 receptors, but enhanced by the absence of CysLT2 receptors. CysLT1 and CysLT2 receptors colocalized to the plasma membranes and nuclei of a human MC line, LAD2. Antibody-based fluorescent lifetime imaging microscopy confirmed complexes containing both receptors based on fluorescence energy transfer. Negative regulation of CysLT1-induced mitogenic signaling responses of MCs by CysLT2 demonstrates physiologically relevant functions for GPCR heterodimers on primary cells central to inflammation.

Do orphan G‐protein‐coupled receptors have ligand‐independent functions?

G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observation that orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by so doing regulate the function of the latter. Pairing orphan GPCRs with their potential heterodimerization partners will have a major impact on our understanding of the extraordinary diversity offered by GPCR heterodimerization and, in addition, will constitute a novel strategy to elucidate the function of orphan receptors that needs to be added to the repertoire of 'deorphanization' strategies.

Heterodimeric adenosine receptors: a device to regulate neurotransmitter release.

Since 1990 it has been known that dimers are the basic functional form of nearly all G-protein-coupled receptors (GPCRs) and that homo- and heterodimerization may play a key role in correct receptor maturation and trafficking to the plasma membrane. Nevertheless, homo- and heterodimerization of GPCR has become a matter of debate especially in the search for the precise physiological meaning of this phenomenon. This article focuses on how heterodimerization of adenosine A1 and A2A receptors, which are coupled to apparently opposite signalling pathways, allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release.

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.

Receptor heterodimerization: a new level of cross-talk

Most G protein-coupled receptors (GPCRs) probably exist as homodimers, but it is increasingly recognized that GPCRs may also dimerize with other types of GPCRs and that this physical interaction may affect the function of either receptor. A study in this issue of the JCI demonstrates how heterodimerization between prostaglandin E receptors and beta(2)-adrenergic receptors (beta(2)ARs) in airway smooth muscle cells results in uncoupling of beta(2)ARs and a diminished bronchodilator response to beta(2)AR agonists (see the related article beginning on page 1400). This illustrates what we believe to be a novel mechanism of receptor cross-talk and highlights the potential importance of GPCR heterodimerization in diseases such as asthma and how this could lead to the development of more specific therapies in the future.

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.

The quaternary structure of G protein‐coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane-helix polypeptides that are encoded by somewhere in the region of 3% of the genes of mammalian genomes and are the most actively targeted class of polypeptides for small molecule drug design. Despite this, and observations scattered throughout the literature for a number of years suggesting that GPCRs can exist as dimers or higher-order oligomers, it is only in the last five years that analysis of the structural organization of GPCRs has become wide-ranging. Although a number of key issues, including the molecular basis of dimerization ⁄oligomerization, the importance of dimerization ⁄oligomerization for function, and the physiological occurrence and relevance of hetero-dimers containing GPCRs produced by two different genes, remain both contentious and central research themes, there is now little doubt that dimerization ⁄oligomerization does occur. The four minireviews in this issue of FEBS Journal address different aspects of GPCR dimerization ⁄oligomerization. In the first of these Michel Bouvier and I discuss, and address critically, the approaches that have and are being employed to examine GPCR dimerization ⁄ oligomerization. Given the large number of studies that are being performed in this area and the limitations of each individual technique we conclude that concurrent application of a range of approaches that employ distinct techniques is required to provide convincing data. At the current time experimental information on the mechanisms of dimerization and the interfaces involved is both limited and fragmentary. Indeed, models for dimerization include both ‘linear packing’ models in which the external faces of specific transmembrane helices provide the interface and ‘domain swap’ models in which the two elements of the dimer exchange transmembrane segments. Informatics can suggest plausible mechanisms and in the second article Marta Filizola and Harel Weinstein consider the contributions that informatic studies have made to this field and the manner in which iterative cycles of informatic analysis and direct experiments may lead to clearer understanding. The third article by Roberto Maggio and colleagues focuses on the development of distinct pharmacology and function associated with GPCR hetero-dimer formation. As noted above, there is considerable interest in the possibility that at least a proportion of GPCR ‘hetero-dimers’ represent ‘domain swaps’. In such a situation it is almost axiomatic that differences in the details of ligand binding would be produced in comparison with ‘linear packing’ dimers. The extent of existence of GPCR hetero-dimers in physiological settings remains to be established but if they are relatively common they may represent a novel and currently unappreciated set of drug targets for the pharmaceutical industry. Although the first three articles all concentrate on members of the rhodopsin-like (or family A) GPCRs because numerically these are by far the largest group, the first clear example of GPCR hetero-dimerization and its importance for function came from the family C GPCRs. These all contain a long N-terminal extracellular domain in which the natural ligands bind within a ‘venus flytrap’ structure. Crystal structures of such domains in both empty and ligand-bound arrangements have provided detailed insights into conformational reorganization of such GPCR dimers in the presence of ligands. The ability to create chimeric family C GPCRs by combining the extracellular domain of one with the transmembrane and intracellular element of a second has also provided dramatic insights into the importance of dimerization for function, and in the final article in the series Jean-Phillipe Pin and colleagues discuss such studies and the development of allosteric modulators of function of family C GPCRs, another area with major implications for the development of novel classes of small molecule therapeutics.

Monitoring Receptor Dimerisation

In recent times the concept that G protein-coupled receptors (GPCRs) can exist and potentially function as dimers or higher oligomers has become widely accepted. By considering GPCR-G protein fusion proteins as bi-functional polypeptides we have generated complementary pairs of non-functional mutants that are able to reconstitute function when co-expressed. Importantly for studies on the selectivity of GPCR hetero-dimerisation this allows development of assays in which the hetero-dimer, but not the co-expressed homo-dimers, are functional. The concept that GPCR hetero-dimers display distinct pharmacology can thus be tested directly.

G protein-coupled receptors: Dominant players in cell-cell communication

The G protein-coupled receptors (GPCRs) are the most numerous and the most diverse type of receptors (1-5% of the complete invertebrate and vertebrate genomes). They transduce messages as different as odorants, nucleotides, nucleosides, peptides, lipids, and proteins. There are at least eight families of GPCRs that show no sequence similarities and that use different domains to bind ligands and activate a similar set of G proteins. Homo- and heterodimerization of GPCRs seem to be the rule, and in some cases an absolute requirement, for activation. There are about 100 orphan GPCRs in the human genome which will be used to find new message molecules. Mutations of GPCRs are responsible for a wide range of genetic diseases. The importance of GPCRs in physiological processes is illustrated by the fact that they are the target of the majority of therapeutical drugs and drugs of abuse.

Structural models for dimerization of G‐protein coupled receptors: The opioid receptor homodimers

Among the most exciting functional features of G-protein coupled receptors (GPCRs) that are coming into focus lately are those relating to the role and structural characteristics of their oligomerization (mostly homo- and heterodimers). The structural underpinnings of these novel functional insights are still not clear, as current experimental techniques have not yet succeeded in identifying the dimerization interfaces between GPCR monomers. Two computational approaches have recently been designed in our lab to provide reasonable three-dimensional (3D) molecular models of the transmembrane (TM) regions of GPCR dimers based on a combination of the structural information of receptor monomers and analyses of correlated mutations in receptor families. The modeling of GPCR heterodimers has been described recently. We present here a related approach for modeling of GPCR homodimers that identifies the interfaces in the most likely configurations of the complexes. The approach is illustrated for the three cloned opioid receptor subtypes (OPRD, OPRM, and OPRK).

Reply: beyond receptor dimerization

We are grateful to Lakshmi Devi for her thoughtful comments on our recent work and her clear review of the latest publications on the oligomerization of G-protein-coupled receptors (GPCRs). As Devi mentioned, the use of resonance energy transfer techniques to study homo- or heterodimerization of different GPCRs has provided the first direct experimental evidence for the existence of receptor oligomers in living cells.