Pericardial Adipose Tissue Regulates Granulopoiesis, Fibrosis, and Cardiac Function After Myocardial Infarction.

BACKGROUND: The pericardial adipose tissue (AT) contains a high density of lymphoid clusters. It is unknown whether these clusters play a role in post-myocardial infarction (MI) inflammatory responses and cardiac outcome. METHODS: Lymphoid clusters were examined in epicardial AT of humans with or without coronary artery disease. Murine pericardial lymphoid clusters were visualized in mice subjected to coronary artery ligation. To study the relevance of pericardial clusters during inflammatory responses after MI, we surgically removed the pericardial AT and performed B-cell depletion and granulocyte-macrophage colony-stimulating factor blockade. Leukocytes in murine hearts, pericardial AT, spleen, mediastinal lymph nodes, and bone marrow were quantified by flow cytometry. Cannabinoid receptor CB2 (CB2-/-) mice were used as a model for enhanced B-cell responses. The effect of impaired dendritic cell (DC) trafficking on pericardial AT inflammatory responses was tested in CCR7-/- mice subjected to MI. Cardiac fibrosis and ventricular function were assessed by histology and echocardiography. RESULTS: We identified larger B-cell clusters in epicardial AT of human patients with coronary artery disease in comparison with controls without coronary artery disease. Infarcted mice also had larger pericardial clusters and 3-fold upregulated numbers of granulocyte-macrophage colony-stimulating factor-producing B cells within pericardial AT, but not spleen or lymph nodes. This was associated with higher DC and T-cell counts in pericardial AT, which outnumbered DCs and T cells in lymph nodes. Analysis of DC maturation markers, tracking experiments with fluorescently labeled cells, and use of CCR7-deficient mice suggested that activated DCs migrate from infarcts into pericardial AT via CCR7. B-cell depletion or granulocyte-macrophage colony-stimulating factor neutralization inhibited DC and T-cell expansion within pericardial AT, and translated into reduced bone marrow granulopoiesis and cardiac neutrophil infiltration 3 days after MI. The relevance of the pericardial AT in mediating all these effects was confirmed by removal of pericardial AT and ex vivo coculture with pericardial AT and granulocyte progenitors. Finally, enhanced fibrosis and worsened ejection fraction in CB2-/- mice were limited by pericardial AT removal. CONCLUSIONS: Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.

Partial Agonist and Biased Signaling Properties of the Synthetic Enantiomers J113863/UCB35625 at Chemokine Receptors CCR2 and CCR5.

Biased agonism at G protein-coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12 Binding of J113863 to CCR2 or CCR5 also induced the recruitment of ß-arrestin 2, whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and ß-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist, or full agonist according to the receptor, the enantiomer, and the signaling pathway investigated.

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32.

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.

Biased signaling at chemokine receptors.

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as ß-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gαi subtypes (Gαi1, Gαi2, and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα12, but not Gα13. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.

The Atypical Chemerin Receptor GPR1 Displays Different Modes of Interaction with ß-Arrestins in Humans and Mice with Important Consequences on Subcellular Localization and Trafficking.

Atypical chemokine receptors (ACKRs) have emerged as a subfamily of chemokine receptors regulating the local bioavailability of their ligands through scavenging, concentration, or transport. The biological roles of ACKRs in human physiology and diseases are often studied by using transgenic mouse models. However, it is unknown whether mouse and human ACKRs share the same properties. In this study, we compared the properties of the human and mouse atypical chemerin receptor GPR1 and showed that they behave differently regarding their interaction with ß-arrestins. Human hGPR1 interacts with ß-arrestins as a result of chemerin stimulation, whereas its mouse orthologue mGPR1 displays a strong constitutive interaction with ß-arrestins in basal conditions. The constitutive interaction of mGPR1 with ß-arrestins is accompanied by a redistribution of the receptor from the plasma membrane to early and recycling endosomes. In addition, ß-arrestins appear mandatory for the chemerin-induced internalization of mGPR1, whereas they are dispensable for the trafficking of hGPR1. However, mGPR1 scavenges chemerin and activates MAP kinases ERK1/2 similarly to hGPR1. Finally, we showed that the constitutive interaction of mGPR1 with ß-arrestins required different structural constituents, including the receptor C-terminus and arginine 3.50 in the second intracellular loop. Altogether, our results show that sequence variations within cytosolic regions of GPR1 orthologues influence their ability to interact with ß-arrestins, with important consequences on GPR1 subcellular distribution and trafficking.

Significance of N-terminal proteolysis of CCL14a to activity on the chemokine receptors CCR1 and CCR5 and the human cytomegalovirus-encoded chemokine receptor US28.

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1-74) circulates in high concentrations in plasma. CCL14a(1-74) is converted into CCL14a(9-74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12-74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The rank order of binding affinity to these receptors and of the activation of CCR1 and CCR5-mediated intracellular Ca(2+) concentration mobilization is CCL14a(6-74)<(7-74)<(8-74)<<(9-74) = (10-74)>>(11-74)>>(12-74). The almost identical affinities of CCL14a(7-74), CCL14a(9-74), and CCL14a(10-74) for the US28 receptor and the inhibition of US28-mediated HIV infection of 293T cells by all of the N-terminally truncated CCL14a analogs support the promiscuous nature of the viral chemokine receptor US28. In high concentrations, CCL14a(12-74) did reveal antagonistic activity on intracellular Ca(2+) concentration mobilization in CCR1- and CCR5-transfected cells, which suggests that truncation of Tyr(11) might be of significance for an efficient inactivation of CCL14a. A putative inactivation pathway of CCL14a(9-74) to CCL14a(12-74) may involve the dipeptidase CD26/dipeptidyl peptidase IV (DPPIV), which generates CCL14a(11-74), and the metalloprotease aminopeptidase N (CD13), which displays the capacity to generate CCL14a(12-74) from CCL14a(11-74). Our results suggest that the activity of CCL14a might be regulated by stringent proteolytic activation and inactivation steps.

TAFA4 relieves injury-induced mechanical hypersensitivity through LDL receptors and modulation of spinal A-type K+ current.

Pain, whether acute or persistent, is a serious medical problem worldwide. However, its management remains unsatisfactory, and new analgesic molecules are required. We show here that TAFA4 reverses inflammatory, postoperative, and spared nerve injury (SNI)-induced mechanical hypersensitivity in male and female mice. TAFA4 requires functional low-density lipoprotein receptor-related proteins (LRPs) because their inhibition by RAP (receptor-associated protein) dose-dependently abolishes its antihypersensitive actions. SNI selectively decreases A-type K+ current (IA) in spinal lamina II outer excitatory interneurons (L-IIo ExINs) and induces a concomitant increase in IA and decrease in hyperpolarization-activated current (Ih) in lamina II inner inhibitory interneurons (L-IIi InhINs). Remarkably, SNI-induced ion current alterations in both IN subtypes were rescued by TAFA4 in an LRP-dependent manner. We provide insights into the mechanism by which TAFA4 reverses injury-induced mechanical hypersensitivity by restoring normal spinal neuron activity and highlight the considerable potential of TAFA4 as a treatment for injury-induced mechanical pain.

Hetero-oligomerization of CCR2, CCR5, and CXCR4 and the protean effects of "selective" antagonists.

Chemokine receptors constitute an attractive family of drug targets in the frame of inflammatory diseases. However, targeting specific chemokine receptors may be complicated by their ability to form dimers or higher order oligomers. Using a combination of luminescence complementation and bioluminescence resonance energy transfer assays, we demonstrate for the first time the existence of hetero-oligomeric complexes composed of at least three chemokine receptors (CCR2, CCR5, and CXCR4). We show in T cells and monocytes that negative binding cooperativity takes place between the binding pockets of these receptors, demonstrating their functional interaction in leukocytes. We also show that specific antagonists of one receptor (TAK-779 or AMD3100) lead to functional cross-inhibition of the others. Finally, using the air pouch model in mice, we show that the CCR2 and CCR5 antagonist TAK-779 inhibits cell recruitment promoted by the CXCR4 agonist SDF-1 alpha, demonstrating that cross-inhibition by antagonists also occurs in vivo. Thus, antagonists of the therapeutically important chemokine receptors regulate the functional properties of other receptors to which they do not bind directly with important implications for the use of these agents in vivo.

Chemerin Added to Endothelin-1 Promotes Rat Pulmonary Artery Smooth Muscle Cell Proliferation and Migration.

BACKGROUND: While chemerin has been shown to increase proliferation and migration of systemic vascular smooth muscle cells (SMCs) contributing therefore to the development of hypertension, this remains to be clarified for the pulmonary circulation. METHODS: Expression of chemerin and its three receptors (CMKRL1, CCRL2, GPR1) was examined by immunohistochemistry and RTq-PCR in lungs, pulmonary artery, and thoracic aorta from Wistar rats. Primary cultured rat pulmonary artery and thoracic aorta SMCs treated with recombinant chemerin (tested from 5.10-9 to 10-7 mol/L) were assessed for proliferation and migration (both with 10-7 mol/L endothelin-1), as well as for staurosporine-induced apoptosis. RESULTS: In pulmonary artery and thoracic aorta, CMKLR1 expression was detected in both endothelial cells and SMCs. In primary cultured pulmonary artery SMCs, chemerin and its three receptors were expressed, and CMKLR1 expression was higher than those of CCRL2 and GPR1. Chemerin added to endothelin-1 increased pulmonary artery SMC proliferation, while chemerin or endothelin-1 alone did not. This effect was less pronounced in thoracic aorta SMCs. Chemerin induced pulmonary artery and thoracic aorta SMC migration, which was exacerbated by endothelin-1 and more pronounced in thoracic aorta SMCs. Chemerin concentration-dependently reduced staurosporine-induced apoptosis in both pulmonary artery and thoracic aorta SMCs. In pulmonary artery SMCs, endothelin-1 treatment increased the expression of CMKLR1, CCRL2, and GPR1, while these expressions were not altered in thoracic aorta SMCs. CONCLUSION: Chemerin/CMKRL1 signaling, in conjunction with a key mediator in the pathogenesis of pulmonary hypertensive diseases, endothelin-1, stimulated proliferation and migration, and increased resistance to apoptosis in rat primary cultured pulmonary artery SMCs. Our results suggest that this signaling could play a role in pulmonary artery remodeling observed in pulmonary hypertension.

Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor?

G protein-coupled receptors (GPCRs) are membrane-bound receptors that are considered prime candidates for the development of novel insect pest management strategies. However, the molecular signaling properties of insect GPCRs remain poorly understood. In fact, most studies on insect GPCR signaling are limited to analysis of fluctuations in the secondary messenger molecules calcium (Ca2+) and/or cyclic adenosine monophosphate (cAMP). In the current study, we characterized a corticotropin-releasing factor-related diuretic hormone (CRF-DH) receptor of the desert locust, Schistocerca gregaria. This Schgr-CRF-DHR is mainly expressed in the nervous system and in brain-associated endocrine organs. The neuropeptide Schgr-CRF-DH induced Ca2+-dependent aequorin-based bioluminescent responses in CHO cells co-expressing this receptor with the promiscuous Gα16 protein. Furthermore, when co-expressed with the cAMP-dependent bioluminescence resonance energy transfer (BRET)-based CAMYEL biosensor in HEK293T cells, this receptor elicited dose-dependent agonist-induced responses with an EC50 in the nanomolar range (4.02 nM). In addition, we tested if vertebrate BRET-based G protein biosensors, can also be used to detect direct Gα protein subunit activation by an insect GPCR. Therefore, we analyzed ten different human BRET-based G protein biosensors, representing members of all four Gα protein subfamilies; Gαs, Gαi/o, Gαq/11 and Gα12/13. Our data demonstrate that stimulation of Schgr-CRF-DHR by Schgr-CRF-DH can dose-dependently activate Gαi/o and Gαs biosensors, while no significant effects were observed with the Gαq/11 and Gα12/13 biosensors. Our study paves the way for future biosensor-based studies to analyze the signaling properties of insect GPCRs in both fundamental science and applied research contexts.

Coexpression of CCR7 and CXCR4 During B Cell Development Controls CXCR4 Responsiveness and Bone Marrow Homing.

The CXCL12-CXCR4 axis plays a key role in the retention of stem cells and progenitors in dedicated bone marrow niches. It is well-known that CXCR4 responsiveness in B lymphocytes decreases dramatically during the final stages of their development in the bone marrow. However, the molecular mechanism underlying this regulation and whether it plays a role in B-cell homeostasis remain unknown. In the present study, we show that the differentiation of pre-B cells into immature and mature B cells is accompanied by modifications to the relative expression of chemokine receptors, with a two-fold downregulation of CXCR4 and upregulation of CCR7. We demonstrate that expression of CCR7 in B cells is involved in the selective inactivation of CXCR4, and that mature B cells from CCR7-/- mice display higher responsiveness to CXCL12 and improved retention in the bone marrow. We also provide molecular evidence supporting a model in which upregulation of CCR7 favors the formation of CXCR4-CCR7 heteromers, wherein CXCR4 is selectively impaired in its ability to activate certain G-protein complexes. Collectively, our results demonstrate that CCR7 behaves as a novel selective endogenous allosteric modulator of CXCR4.

Chemerin influences endothelin- and serotonin-induced pulmonary artery vasoconstriction in rats.

AIMS: Chemerin has been recently identified as a vasoactive adipokine implicated in blood pressure regulation. In this context, we evaluated whether chemerin could influence pulmonary vasoreactive response. MATERIALS AND METHODS: Vascular reactivity to chemerin and to phenylephrine, serotonin and endothelin-1 after chemerin pretreatment was evaluated in rat isolated pulmonary artery versus thoracic aorta with and without endothelium. Vasoreactivity to acetylcholine in presence of nitric oxide (NO)-synthase inhibitor (L-NAME) and to NO donor sodium nitroprusside (SNP) was evaluated in chemerin-pretreated pulmonary artery versus thoracic aorta with endothelium. Pretreatment with ODQ, a soluble guanylate cyclase inhibitor and apocynin, a ROS production inhibitor, were also tested. Arteries and lung tissue were harvested for pathobiological evaluation. KEY FINDINGS: Chemerin contracted endothelium-denuded pulmonary artery, while no response was observed in arteries with endothelium. Chemerin potentiated phenylephrine-, endothelin-1- and serotonin-induced vasoconstriction, which was further enhanced by endothelium removal. Chemerin decreased acetylcholine-induced vasorelaxation in arteries with endothelium, while it did not affect SNP-induced relaxation. In presence of L-NAME, there remained a vasorelaxation in chemerin-pretreated arteries. Chemerin or ODQ alone partly decreased acetylcholine-induced vasorelaxation in pulmonary artery and thoracic aorta, while combined chemerin and ODQ incubation abolished it. Treatment with apocynin partly or totally reversed chemerin effects. In both types of arteries, chemerin reduced acetylcholine-induced NO production, as well as endothelial and inducible NO-synthase expression. SIGNIFICANCE: Chemerin potentiates vascular responses to vasoconstrictors in pulmonary artery and thoracic aorta and, impairs acetylcholine-induced pulmonary artery vasodilatation, by mechanisms involving at least partly NO signaling and oxidative stress.

Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events.

CC and CXC chemokines coinduced in fibroblasts and leukocytes by cytokines and microbial agents determine the number of phagocytes infiltrating into inflamed tissues. Interleukin-8/CXCL8 and stromal cell-derived factor-1/CXCL12 significantly and dose-dependently increased the migration of monocytes, expressing the corresponding CXC chemokine receptors CXCR2 and CXCR4, toward suboptimal concentrations of the monocyte chemotactic proteins CCL2 or CCL7. These findings were confirmed using different chemotaxis assays and monocytic THP-1 cells. In contrast, the combination of two CC chemokines (CCL2 plus CCL7) or two CXC chemokines (CXCL8 plus CXCL12) did not provide synergy in monocyte chemotaxis. These data show that chemokines competing for related receptors and using similar signaling pathways do not synergize. Receptor heterodimerization is probably not essential for chemokine synergy as shown in CXCR4/CCR2 cotransfectants. It is noteworthy that CCL2 mediated extracellular signal-regulated kinase 1/2 phosphorylation and calcium mobilization was significantly enhanced by CXCL8 in monocytes, indicating cooperative downstream signaling pathways during enhanced chemotaxis. Moreover, in contrast to intact CXCL12, truncated CXCL12(3-68), which has impaired receptor signaling capacity but can still desensitize CXCR4, was unable to synergize with CCL2 in monocytic cell migration. Furthermore, AMD3100 and RS102895, specific CXCR4 and CCR2 inhibitors, respectively, reduced the synergistic effect between CCL2 and CXCL12 significantly. These data indicate that for synergistic interaction between chemokines binding and signaling of the two chemokines via their proper receptors is necessary.

Highly potent HIV inhibition: engineering a key anti-HIV structure from PSC-RANTES into MIP-1 beta/CCL4.

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1beta/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1beta/CCL4 analogues that retain the receptor binding profile of MIP-1beta/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potency.

Allosteric properties of G protein-coupled receptor oligomers.

Allosteric regulation of ligand binding is a well-established mechanism regulating the function of G protein-coupled receptors (GPCR). Allosteric modulators have been considered so far as molecules binding to an allosteric site, distinct from that of the reference ligand (orthosteric site), and able to modulate the binding affinity at the orthosteric site and/or the signaling properties resulting from orthosteric site occupancy. Given that most GPCR are known to form dimers or higher order oligomers, we explored whether allosteric interactions could also occur between protomers within oligomeric arrays, thereby influencing binding and signaling receptor properties. Two main conclusions emerged from such studies. First, allosteric modulators can affect one receptor by binding to another receptor within a dimeric or oligomeric complex. Second, allosteric modulators might act on a given receptor by targeting the "orthosteric site" in another receptor of the complex. Allosteric regulation within di(oligo)mers thus implies that the pharmacological properties of a given receptor subtype can be influenced by the array of dimerization partners coexpressed in each particular cell type. Ligands could thus act as agonists or antagonists on 1 receptor, while modulating allosterically the function of a variety of other receptors to which they do not bind directly. Allosteric regulation across GPCR oligomeric interfaces is expected to greatly influence the practice of pharmacology. It will likely affect the design of drug discovery programs, which rely mostly on the overexpression of the receptor of interest in a cell line, thereby focusing on homo-oligomers and ignoring the potential effects of other partners.

The activation mechanism of chemokine receptor CCR5 involves common structural changes but a different network of interhelical interactions relative to rhodopsin.

In G protein-coupled receptors (GPCRs), the interaction between the cytosolic ends of transmembrane helix 3 (TM3) and TM6 was shown to play an important role in the transition from inactive to active states. According to the currently prevailing model, constructed for rhodopsin and structurally related receptors, the arginine of the conserved "DRY" motif located at the cytosolic end of TM3 (R3.50) would interact with acidic residues in TM3 (D/E3.49) and TM6 (D/E6.30) at the resting state and shift out of this polar pocket upon agonist stimulation. However, 30% of GPCRs, including all chemokine receptors, contain a positively charged residue at position 6.30 which does not support an interaction with R3.50. We have investigated the role of R6.30 in this receptor family by using CCR5 as a model. R6.30D and R6.30E substitutions, which allow an ionic interaction with R3.50, resulted in an almost silent receptor devoid of constitutive activity and strongly impaired in its ability to bind chemokines but still able to internalize. R6.30A and R6.30Q substitutions, allowing weaker interactions with R3.50, preserved chemokine binding but reduced the constitutive activity and the functional response to chemokines. These results indicate that the constitutive and ligand-promoted activity of CCR5 can be modified by modulating the interaction between the DRY motif in TM3 and residues in TM6 suggesting that the overall structure and activation mechanism are well conserved in GPCRs. However, the molecular interactions locking the inactive state must be different in receptors devoid of D/E6.30.

Dimerization of chemokine receptors and its functional consequences.

It became clear over the recent years that most, if not all, G protein-coupled receptors (GPCR) are able to form dimers or higher order oligomers. Chemokine receptors make no exception to this new rule and both homo- and heterodimerization were demonstrated for CC and CXC receptors. Functional analyses demonstrated negative binding cooperativity between the two subunits of a dimer. The consequence is that only one chemokine can bind with high affinity onto a receptor dimer. In the context of receptor activation, this implies that the motions of helical domains triggered by the binding of agonists induce correlated changes in the other protomer. The impact of the chemokine dimerization process in terms of co-receptor function and drug development is discussed.

Identification of nonpeptide CCR5 receptor agonists by structure-based virtual screening.

A three-dimensional model of the chemokine receptor CCR5 has been built to fulfill structural peculiarities of its alpha-helix bundle and to distinguish known CCR5 antagonists from randomly chosen drug-like decoys. In silico screening of a library of 1.6 million commercially available compounds against the CCR5 model by sequential filters (drug-likeness, 2-D pharmacophore, 3-D docking, scaffold clustering) yielded a hit list of 59 compounds, out of which 10 exhibited a detectable binding affinity to the CCR5 receptor. Unexpectedly, most binders tested in a functional assay were shown to be agonists of the CCR5 receptor. A follow-up database query based on similarity to the most potent binders identified three new CCR5 agonists. Despite a moderate affinity of all nonpeptide ligands for the CCR5 receptor, one of the agonists was shown to promote efficient receptor internalization, which is a process therapeutically favorable for protection against HIV-1 infection.

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of ß-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate ß-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and ß-arrestin2 activation but not ß-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking.

The membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control. Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole. This down-regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins. Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking. We show that Npi3 is required for NH4+-induced down-regulation of Gap1, and particularly for efficient ubiquitination of the permease. Npi3 plays a pleiotropic role in permease down-regulation, since it is also involved in ubiquitination and stress-induced down-regulation of the uracil permease Fur4 and in glucose-induced degradation of hexose transporters Hxt6/7. We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells. NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover. Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis. We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin-dependent control of membrane protein trafficking.