Emerging Roles of the Atypical Chemokine Receptor 3 (ACKR3) in Cardiovascular Diseases.

Chemokines, and their receptors play a crucial role in the pathophysiology of cardiovascular diseases (CVD). Chemokines classically mediate their effects by binding to G-protein-coupled receptors. The discovery that chemokines can also bind to atypical chemokine receptors (ACKRs) and initiate alternative signaling pathways has changed the paradigm regarding chemokine-related functions. Among these ACKRs, several studies have highlighted the exclusive role of ACKR3, previously known as C-X-C chemokine receptor type 7 (CXCR7), in CVD. Indeed, ACKR3 exert atheroprotective, cardioprotective and anti-thrombotic effects through a wide range of cells including endothelial cells, platelets, inflammatory cells, fibroblasts, vascular smooth muscle cells and cardiomyocytes. ACKR3 functions as a scavenger receptor notably for the pleiotropic chemokine CXCL12, but also as a activator of different pathways such as ß-arrestin-mediated signaling or modulator of CXCR4 signaling through the formation of ACKR3-CXCR4 heterodimers. Hence, a better understanding of the precise roles of ACKR3 may pave the way towards the development of novel and improved therapeutic strategies for CVD. Here, we summarize the structural determinant characteristic of ACKR3, the molecules targeting this receptor and signaling pathways modulated by ACKR3. Finally, we present and discuss recent findings regarding the role of ACKR3 in CVD.

Splenic Marginal Zone B Lymphocytes Regulate Cardiac Remodeling After Acute Myocardial Infarction in Mice.

BACKGROUND: Mature B lymphocytes alter the recovery of cardiac function after acute myocardial infarction (MI) in mice. Follicular B cells and marginal zone B (MZB) cells are spatially distinct mature B-cell populations in the spleen, and they exert specific functional properties. microRNA-21 (miR21)/hypoxia-inducible factor-α (HIF-α)-related pathways have been shown to govern B-cell functions. OBJECTIVES: The goal of this study was to unravel the distinct role of MZB cells and that of endogenous activation of miR21/HIF-α signaling in MZB cells during post-ischemic injury. METHODS: Acute MI was induced in mice by permanent ligation of the left anterior descending coronary artery. Cardiac function and remodeling were assessed by using echocardiography and immunohistochemistry. To determine the specific role of MZB cells, the study used mice with B-cell lineage-specific conditional deletion of Notch signaling, which leads to selection deficiency of MZB cells. To evaluate the role of the HIF-1α isoform, mice were generated with MZB-cell lineage-specific conditional deletion of Hif1a. RESULTS: Acute MI prompted an miR21-dependent increase in HIF-1α, particularly in splenic MZB cells. MZB cell deficiency and MZB cell-specific deletion of miR21 or Hif1a improved cardiac function after acute MI. miR21/HIF-1α signaling in MZB cells was required for Toll-like receptor dependent expression of the monocyte chemoattractant protein CCL7, leading to increased mobilization of inflammatory monocytes to the ischemic myocardium and to adverse post-ischemic cardiac remodeling. CONCLUSIONS: This work reveals a novel function for the miR21/HIF-1α pathway in splenic MZB cells with potential major implications for the modulation of cardiac function after acute MI.

High-Density Lipoprotein Therapy in Stroke: Evaluation of Endothelial SR-BI-Dependent Neuroprotective Effects.

High-density lipoproteins (HDLs) display endothelial protective effects. We tested the role of SR-BI, an HDL receptor expressed by endothelial cells, in the neuroprotective effects of HDLs using an experimental model of acute ischemic stroke. After transient intraluminal middle cerebral artery occlusion (tMCAO), control and endothelial SR-BI deficient mice were intravenously injected by HDLs or saline. Infarct volume and blood-brain barrier (BBB) breakdown were assessed 24 h post tMCAO. The potential of HDLs and the role of SR-BI to maintain the BBB integrity was assessed by using a human cellular model of BBB (hCMEC/D3 cell line) subjected to oxygen-glucose deprivation (OGD). HDL therapy limited the infarct volume and the BBB leakage in control mice relative to saline injection. Interestingly, these neuroprotective effects were thwarted by the deletion of SR-BI in endothelial cells and preserved in mice deficient for SR-BI in myeloid cells. In vitro studies revealed that HDLs can preserve the integrity of the BBB in OGD conditions, and that this effect was reduced by the SR-BI inhibitor, BLT-1. The protection of BBB integrity plays a pivotal role in HDL therapy of acute ischemic stroke. Our results show that this effect is partially mediated by the HDL receptor, SR-BI expressed by endothelial cells.

Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors.

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Low levels of low-density lipoprotein-C associated with proprotein convertase subtilisin kexin 9 inhibition do not increase the risk of hemorrhagic transformation.

BACKGROUND AND PURPOSE: Low levels of low-density lipoprotein-cholesterol (LDL-C) are suspected to be associated with a risk of hemorrhagic transformation after ischemic stroke. We assessed the risk of hemorrhagic transformation after cerebral ischemia/reperfusion in mice with low levels of LDL-C resulting from proprotein convertase subtilisin kexin 9 (PCSK9) deficiency. METHODS: PCSK9-/- and PCSK9+/+ mice were fed with a high-fat/high-cholesterol (21%/0.15%) diet for 1 month. Plasma lipids were measured using colorimetric assays. PCSK9-/- and PCSK9+/+ mice (n=15 per group) were subjected to a 4-hour intraluminal occlusion of the middle cerebral artery followed by 20 hours of reperfusion. Spontaneous hemorrhagic transformation was assessed by quantification of hemoglobin in ischemic tissue. In vitro, a cell model of blood-brain barrier was used to test endothelial barrier integrity in response to decreasing concentrations of LDL-C from 1 to 0.25g/L in ischemia/reperfusion conditions. RESULTS: PCSK9-/- mice had lower LDL-C, high-density lipoprotein-cholesterol, and total cholesterol levels than PCSK9+/+ mice before and after 1 month on the high-fat/high-cholesterol diet. Hemoglobin concentration in ischemic cerebral tissue was not different between PCSK9-/- and PCSK9+/+ mice (31.5 [18.9-60.1] and 32.8 [14.7-69.9] ng/mg protein, respectively; P=0.81). Infarct volume was also similar in both groups (P=0.66). Incubation of human cerebral endothelial cells with decreasing concentrations of LDL-C under ischemia/reperfusion conditions did not alter blood-brain barrier permeability. CONCLUSIONS: Low levels of LDL-C did not increase the risk of hemorrhagic transformation after cerebral ischemia/reperfusion in mice. Our observations suggest that PCSK9 inhibition, leading to LDL-C lowering, should not increase hemorrhagic complications after acute ischemic stroke.

High-density lipoproteins potentiate α1-antitrypsin therapy in elastase-induced pulmonary emphysema.

Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1-3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 µm versus 30.7 ± 4.5 µm; P < 0.001), whereas injection of HDL or AAT alone only showed a moderate, nonsignificant protective effect (28.2 ± 4.2 µm versus 30.7 ± 5 µm [P = 0.23] and 27.3 ± 5.66 µm versus 30.71 ± 4.96 µm [P = 0.18], respectively). Indeed, protection by HDL-AAT was significantly higher than that observed with HDL or AAT (P = 0.006 and P = 0.048, respectively). This protective effect was associated (at 6, 24, and 72 h) with: (1) a reduction in neutrophil and macrophage number in the bronchoalveolar lavage fluid; (2) decreased concentrations of IL-6, monocyte chemoattractant protein-1, and TNF-α in both bronchoalveolar lavage fluid and plasma; (3) a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-9 activities; and (4) a reduction in the degradation of fibronectin, a marker of tissue damage. In addition, HDL-AAT reduced acute cigarette smoke-induced inflammatory response. Intravenous HDL-AAT treatment afforded a better protection against elastase-induced pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency.

Complementary methods provide evidence for the expression of CXCR7 on human B cells.

PTMs of extracellular domains of membrane proteins can influence antibody binding and give rise to ambivalent results. Best proof of protein expression is the use of complementary methods to provide unequivocal evidence. CXCR7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL12 and CXCL11. The expression of CXCR7 on nonhematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was recently challenged. To solve the dissent, we thoroughly analyzed the expression of CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. Examination of CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human B cells. Moreover, real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally, two CXCR7-specific peptides were identified by MS in immunoprecipitates from primary human B cells. Thus, we present a strategy based on combined proteomic and functional approaches that can be used to solve dissents on protein expression, i.e. demonstrating the expression of CXCR7 on human leukocytes.

Evidence for leptin receptor isoforms heteromerization at the cell surface.

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.

The stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger G(alphai) protein-dependent signaling, although energy transfer assays indicate that it constitutively interacts with G(alphai) proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/G(alphai) protein complexes and impairs CXCR4-promoted G(alphai)-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12.

The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins.

The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor.

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

CXCR4 dimerization and beta-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of CXC chemokine receptor 4 (CXCR4). Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immunohematologic clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on beta-arrestin (betaarr) recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced betaarr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4(1013) maintains association with betaarr2 and triggers augmented and prolonged betaarr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4(1013)-mediated chemotaxis critically requires betaarr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4(1013) abrogates betaarr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4(1013) spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between betaarr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12.

Leukocyte analysis from WHIM syndrome patients reveals a pivotal role for GRK3 in CXCR4 signaling.

Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF (WHIM(WT)) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did WHIM(WT) cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIM(WT) leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIM(WT) patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related WHIM(WT) disorder.

Alternative drug discovery approaches for orphan GPCRs.

G protein-coupled receptors (GPCRs) are well-known drug targets. However, a question mark remains for the more than 100 orphan GPCRs as current deorphanisation strategies failed to identify specific ligands for these receptors. Recent advances have shown that orphan GPCRs may have important functions that are ligand-independent. Orphan GPCRs can modulate the function of well-defined drug targets such as GPCRs with identified ligands and neurotransmitter transporters though physical association with those molecules. Thus, compounds that bind to orphan GPCRs and allosterically regulate the function of the interacting partner or even disrupt the interaction with the latter could become new drugs.

[GPCRs heterodimerization: a new way towards the discovery of function for the orphan receptors?]. / L'hétérodimérisation des récepteurs couplés aux protéines G : une nouvelle voie vers la découverte de fonctions pour les récepteurs orphelins ?

G protein-coupled receptors (GPCRs), also called seven transmembrane domain (7TM) proteins, represent the largest family of cell surface receptors. GPCRs control a variety of physiological processes, are involved in multiple diseases and are major drug targets. Despite a vast effort of academic and industrial research, more than one hundred receptors remain orphans. These orphan GPCRs offer a great potential for drug discovery, as almost 60% of currently prescribed drugs target GPCRs. Deorphenization strategies have concentrated mainly on the identification of the natural ligands of these proteins. Recent advances have shown that orphan GPCRs, similar to orphan nuclear receptors, can regulate the function of non-orphan receptors by heterodimerization. These findings not only help to better understand the extraordinary diversity of GPCRs, but also open new perspectives for the identification of the function of these orphan receptors that hold great therapeutic potential.

Detection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopes.

GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT(1) and MT(2) receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein.

Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers.

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.

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization.

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

Are G protein-coupled receptor heterodimers of physiological relevance?--Focus on melatonin receptors.

In mammals, the circadian hormone melatonin targets two seven-transmembrane-spanning receptors, MT1 and MT2, of the G protein-coupled receptor (GPCR) super-family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT1 and MT2 homodimers and MT1/MT2 heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT1/MT2 heterodimers remains to be shown in native tissues but is suggested by the documented co-expression of MT1 and MT2 in many melatonin-sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.