We have cloned and expressed a novel human Gprotein-coupled receptor closely related to the human P2Y 12 receptor. It corresponds to the orphan receptor called GPR86. GPR86 proved to be a G i -coupled receptor displaying a high affinity for ADP, similar to the P2Y 12 receptor and can therefore be tentatively called P2Y 13 . In 1321N1 cells, the P2Y 13 receptor coupled to the phosphoinositide pathway only when coexpressed with G␣ 16 . Inositol trisphosphate formation was stimulated equipotently by nanomolar concentrations of ADP and 2MeSADP, whereas 2MeSATP and ATP were inactive. In CHO-K1 cells expressing the P2Y 13 receptor, ADP and 2MeSADP had a biphasic effect on the forskolin-stimulated accumulation of cAMP: inhibition at nanomolar concentrations and potentiation at micromolar levels. In the same cells, ADP and 2MeSADP also stimulated the phosphorylation of Erk1 and Erk2, in a pertussis toxinsensitive way. The tissue distribution of P2Y 13 was investigated by reverse transcriptase-polymerase chain reaction, and the predominant signals were obtained in spleen and brain. Although these can be discriminated by tissue distribution and some pharmacological features, the P2Y 12 and P2Y 13 receptors form a subgroup of related P2Y subtypes that is structurally different from the other P2Y subtypes but share coupling to G i and a high affinity for ADP.Adenine and uridine nucleotides induce pharmacological and physiological responses through several G-protein-coupled receptors (P2Y) and ligand-gated cation channels (P2X) (1, 2). The P2Y family encompasses two selective purinoceptors: the human P2Y 1 and P2Y 11 receptors, which are preferentially activated respectively by ADP and ATP (3-5). Nucleotide receptors responsive to both adenine and uracil nucleotides are the P2Y 2 receptor, activated equipotently by ATP and UTP (6, 7) as well as the Xenopus P2Y 8 (8) and turkey tp2y receptor (9), activated equally by all triphosphate nucleotides. There are also pyrimidinoceptors: the chicken P2Y 3 (10) and human P2Y 6 (11-13) receptors activated preferentially by UDP, and the human P2Y 4 receptor (13-15) activated preferentially by UTP. All these P2Y subtypes are coupled to the phosphoinositide pathway. The P2Y 11 and tp2y receptors are additionally coupled respectively to stimulation and inhibition of adenylyl cyclase. Other receptors (P2Y 5 , Ref. 16; P2Y 7 , Ref. 17; P2Y 9 , and P2Y 10 ) have been mistakenly included in the P2Y family (18 -20). Recently, a P2Y 12 subtype has been cloned, which corresponds in fact to the platelet ADP receptor previously called P 2T (21,22). It is coupled to an inhibition of adenylyl cyclase and is specifically expressed in the platelets and the brain. Its primary structure is not related to the other P2Y receptors but rather to that of the UDP-glucose receptor (23). Here we report the cloning and characterization of a novel G-protein-coupled receptor that is structurally related to the P2Y 12 receptor and was recently described as an orphan receptor called GPR86 (24).

The P2Y 13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y 12 receptor as well as similar functional properties: coupling to G i and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y 13 receptor and its differences with that of the P2Y 12 Similarly, 2MeSADP was more potent than ADP in stimulating IP 3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP 3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP 3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y 13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y 13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2Ј,4Јdisulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5Ј-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N 6

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).

Extracellular adenosine triphosphate affects the maturation of human monocytederived dendritic cells (DCs), mainly by inhibiting T-helper 1 (Th1) cytokines, promoting Th2 cytokines, and modulating the expression of costimulatory molecules. In this study, we report that adenosine triphosphate (ATP) can induce immunosuppression through its action on DCs, defining a new role for extracellular nucleotides. Microarray analysis of ATPstimulated human DCs revealed inter alia a drastic up-regulation of 2 genes encoding mediators involved in immunosuppression: thrombospondin-1 (TSP-1) and indoleamine 2,3-dioxygenase (IDO). The release of TSP-1 by DCs in response to ATP was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA), immunodetection, and mass spectrometry analysis, and has an antiproliferative effect on T CD4 ؉ lymphocytes through TSP-1/CD47 interaction. Our pharmacologic data support the involvement of purinergic receptor P2Y 11 in this ATP-mediated TSP-1 secretion. We demonstrate also that ATP significantly potentiates the up-regulation of IDO-a negative regulator of T lymphocyte proliferation-and kynurenine production initiated by interferon-␥ (IFN-␥) in human DCs.Thus, extracellular ATP released from damaged cells and previously considered as a danger signal is also a potent regulator of mediators playing key roles in immune tolerance. Consequently, nucleotides' derivatives may be considered as useful tools for DC-based immunotherapies. ( IntroductionDendritic cells (DCs) are antigen-presenting cells (APCs) playing a crucial role in the induction and regulation of immune responses. In response to danger signals like proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤); pathogen-related molecules (lipopolysaccharide [LPS], doublestranded RNA, bacterial DNA); and T cell-derived signals such as CD40 ligand, immature dendritic cells undergo maturation. Maturation of DCs induces the loss of endocytosis, the surface expression of stable major histocompatibility complex (MHC)-peptide complexes and costimulatory molecules (CD80, CD86) and the production of cytokines such as IL-12. 1 Maturation is also accompanied by a shift in the expression of chemokines and their receptors, allowing DC migration to lymphoid organs. High secretion of IL-12 by mature DCs induces differentiation of CD4 ϩ T cells into T-helper 1 (Th1) cells secreting interferon-␥ (IFN-␥), whereas low IL-12 release combined with IL-10 production induces a Th2 response or a T regulatory response associated with induced tolerance. 1,2 In the past several years, our concepts about the role of DCs as the most potent initiators of the immune response against foreign antigens have evolved and the existence of an intermediate state between immaturity and maturation, called semimaturation, has been proposed. 3 An increasing number of reports provide strong evidence that APCs, and particularly DCs, are also involved in central and peripheral tolerance. 4,5 This tolerogenicity seems to be mediated b...

Adenosine 5'-triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up-regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto-enzyme CD39, which converts ATP into adenosine 5'-diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal-regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)-12, tumor necrosis factor alpha (TNF-alpha), IL-10] were investigated in human monocyte-derived DC. ADP, 2-methylthio-ADP, and ADPbetaS had no effect on cAMP, increased [Ca2+]i, and stimulated the phosphorylation of ERK1. The effect on ERK1 was inhibited by AR-C69931MX, a P2Y12 and P2Y13 antagonist. On the contrary the effect on [Ca2+]i was neither inhibited by AR-C69931MX or by the P2Y1 antagonist MRS-2179. Both effects were inhibited by pertussis toxin. ADPbetaS alone was less potent for up-regulation of CD83 than ATPgammaS and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATPgammaS, ADPbetaS inhibited the release of IL-12p40, IL-12p70, and TNF-alpha stimulated by LPS (1-100 ng/ml). The inhibitory effect of ADPbetaS on IL-12 release was neither reversed by AR-C69931MX or by MRS-2179. The two nucleotides had opposite effects on IL-10 production: inhibition by ADPbetaS and potentiation by ATPgammaS. In conclusion, ATP can modulate the function of DC, directly via a cAMP increase mediated by the P2Y11 receptor and indirectly via its degradation into ADP, which acts via Gi-coupled receptors coupled to ERK activation and calcium mobilization. These distinct mechanisms converge on the inhibition of inflammatory cytokine production, particularly IL-12, but have a differential effect on IL-10.