Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 M) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca 2؉ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxinsensitive G i/o family, whereas GPR43 displayed a dual coupling through G i/o and Pertussis toxin-insensitive G q protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca 2؉ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.

When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPl1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPl1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin-protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca(2+)-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPl1/RSP5 product. Chromosomal deletion of NPl1/RSP5 showed that this gene is essential for cell viability. In the viable npi1/rsp5 strain, expression of NPl1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4 + induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in thenpi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4 +-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4 + to mutants defective in endocytosis.

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 β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-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 β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

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␣, 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.Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation and from viral infection to cancer (1, 2). They mediate their effects by binding to cell surface receptors, which belong to the large family of G protein-coupled receptors (GPCRs). 4 Receptor binding initiates a cascade of intracellular events mediated by the association of the receptor with a heterotrimeric G protein, promoting guanine nucleotide exchange in the ␣ subunit and activation of the G protein (3). The G proteins trigger various effector enzymes, leading to chemotaxis and the regulation of a wide range of other functions, which vary in different cell populations. Because of their key role in inflammatory diseases, chemokines and their receptors constitute an attractive family of drug targets. However, the transfer of therapeutic compounds to clinical use has been hampered by the complexity and the functional redundancy of the chemokine system (4). The recent demonstration that chemokine receptors form both homo-and heteromers brings an additional layer of complexity to this system (reviewed in Ref. 5). There is therefore a need for a better understanding of how chemokine receptors are organized and regulated at the supramolecular level in the plasma membrane of primary leukocytes and how this organization affects the activity of agonists and antagonists of these receptors and their subsequent intracellular signaling network. Homodimerization has been reported for four chemokine receptors: CCR2, CCR5, CXCR2, and CXCR4 (6 -12). A recent report suggests that CXCR4 can also form constitutive homooligomers composed of at least three protomers (13). Heteromerization has been demonstrated between CCR2 and CCR5 or...

Chemokine receptors are presently used as targets for candidate drugs in the frame of inflammatory diseases and human immunodeficiency virus infection. They were shown to dimerize, but the functional relevance of dimerization in terms of drug action remains poorly understood. We reported previously the existence of negative binding cooperativity between the subunits of CCR2/CCR5 heterodimers. In the present study, we extend these observations to heterodimers formed by CCR2 and CXCR4, which are more distantly related. We also show that specific antagonists of one receptor inhibit the binding of chemokines to the other receptor as a consequence of their heterodimerization, both in recombinant cell lines and primary leukocytes. This resulted in a significant functional cross-inhibition in terms of calcium mobilization and chemotaxis. These data demonstrate that chemokine receptor antagonists regulate allosterically the functional properties of receptors on which they do not bind directly, with important implications on the effects of these potential therapeutic agents.Chemokine receptors constitute an attractive family of drug targets in the frame of inflammatory diseases, human immunodeficiency virus, and cancer (1, 2). However, the complexity and the functional redundancy of the chemokine system have represented significant hurdles in this development. There is therefore a need for a better comprehension of the chemokine signaling network and how molecules (agonists and antagonists) acting on specific receptors will affect the global function of the chemokine system. One of the challenges in this area is the recent demonstration that G protein-coupled receptors, including chemokine receptors, are able to form dimers, and the functional consequences of this new concept have not been fully appreciated so far.CCR2 is a member of the CC chemokine receptor family that plays an important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury (3). Two isoforms of CCR2 have been described that are generated by alternative splicing and differ in their C-terminal tail, but only the longer variant (CCR2b) is expressed at high level in leukocytes. CCR2 interacts with MCP-1 (CCL2) and other CC chemokines such as MCP-2 (CCL7), MCP-3 (CCL8), and MCP-4 (CCL13) (4). CCR2 was shown to form homodimers, as well as heterodimers with CCR5, its closest structural relative within the chemokine receptor family, resulting in a negative binding cooperativity of allosteric nature (5-8). CCR2 was also reported recently to form heterodimers with CXCR4, a more distantly related chemokine receptor (9). However, no pharmacological characterization of this heterodimer was performed so far. CXCR4 binds specifically SDF-1␣, an evolutionary conserved chemokine known to play a role in a number of physiological processes, such as the homing of T cell populations to sites of inflammation and the maintenance of the cellular microenvironment in bone marrow (10). SDF-1␣ and CXCR4 h...

Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.

It is well established that most G protein-coupled receptors are able to form homo-and heterodimers, although the functional consequences of this process often remain unclear. CCR5 is a chemokine receptor that plays an important role in inflammatory diseases and acts as a major coreceptor for human immunodeficiency viruses. CCR5 was previously shown to homodimerize and heterodimerize with CCR2b, a closely related receptor. In the present study, we have analyzed the functional consequences of this dimerization process, in terms of ligand binding, stimulation of intracellular cascades, and internalization. Bioluminescence resonance energy transfer and coimmunoprecipitation assays demonstrated that CCR5 and CCR2b heterodimerize with the same efficiency as they homodimerize. In contrast to what has been reported previously, no cooperative signaling was observed after costimulation of the two receptors by their respective ligands. However, we observed that CCR5-specific ligands that are unable to compete for monocyte chemoattractant protein (MCP-1) binding on cells expressing CCR2b alone efficiently prevented MCP-1 binding when CCR5 and CCR2b were coexpressed. The extent of this cross-competition was correlated with the amount of CCR5 expressed in cells, as determined by fluorescence-activated cell sorting analysis. Similar observations were made for the CCR2b-selective ligand MCP-1 that competed efficiently for macrophage inflammatory protein-1␤ binding on cells expressing both receptors. Internalization assays did not allow us to demonstrate cointernalization of the receptors in response to agonist stimulation. Together, our observations suggest that CCR5 and CCR2b form homo-and heterodimers with similar efficiencies and that a receptor dimer can only bind a single chemokine.