Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,-and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP 2 hydrolysis, Ca 2؉ mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.

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.

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

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).