Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.

Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular [Ca(2+)] in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of [(3)H]histamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both [(35)S]GTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.

Leukotriene B4 (LTB4) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB4 exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB4 in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB4 elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B4 receptor has been recently identified (LTB4-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB4-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB4-R1. Expression of LTB4-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB4-R2 cDNA displayed high affinity (K d = 0.17 nm) for [3H]LTB4. Radioligand competition assays revealed high affinities of the receptor for LTB4 and LTB5, and 20-hydroxy-LTB4, and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB4 receptor antagonists, 14,15-dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302, had high affinity for LTB4-R1 but not for LTB4-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB4 functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB4 in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBankTM genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC50 = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.