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 (EC 50 ‫؍‬ 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.G-protein coupled receptors (GPCRs) 1 are members of a large protein family that share a common structural motif of seven transmembrane domains (1, 2). GPCRs mediate a variety of physiological functions by interacting with extracellular ligands including small chemical molecules, peptides, and proteins, then transmitting the signal to intracellular second messengers via G proteins (1, 2). In recent years, the primary sequence of many GPCRs have been identified through searching the human genome sequence data base. Whereas these GPCRs have common structural motifs including seven transmembrane domains, often their cognate ligands and biological function are unknown (3-5). To understand the biological functions of these so called orphan receptors, and to enable identification of pharmacological agents active at these receptors, it is helpful to first identify their endogenous cognate ligands (3-5). Currently there are two main approaches generally used to identify orphan receptor ligands. One common approach has been to screen the orphan GPCRs against a collection of known or putative GPCR ligands. This approach has been used successfully to identify several ligand receptor pairings, including MCH, NMU, and P2Y13 etc. (6 -8). However, because ligand collections are somewhat limited, they often do not contain the appropriate ligand for the orphan receptor of interest. Another approach involves purification of the ligands from animal tissue extracts. This approach has also been successful in a number of cases including OFQ/Nociceptin, MCH, Orexin, and P2Y12 etc. (9 -13). However, purification of ligands from animal tissue extracts is labor-intensive and is c...

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 GenBank TM 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 (EC 50 ‫؍‬ 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.

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-R2Melanin-concentrating hormone (MCH) 1 is a cyclic neuropeptide originally identified in salmon pituitaries responsible for skin color changes in teleost fish (1). This peptide is composed of 17 amino acids (DTMRCMVGRVYRPCWEV), whereas the mammalian form of MCH is a 19-amino acid peptide (DFDMLRCMLGRVYRPCWQV) expressed throughout the brain with the highest concentration in the lateral hypothalamic and zona incerta areas (2-4).MCH plays a role in several important physiologic processes.The most active area of research on MCH has focused on the role of MCH in the regulation of feeding behavior and energy balance. Several studies provide evidence for a role for MCH in regulation of body weight.1) Mice that lack MCH are hypophagic and exhibit an increased metabolic rate, resulting in decreased body weight and body fat content (5). 2) Intracerebroventricular injection of MCH increases feeding in rats (6 -8).3) MCH, like several other appetite-regulating neuropeptides, is regulated by leptin in the hypothalamus (9, 10).Other physiological roles of MCH include regulation of the hypothalamopituitary adrenal gland axis (11), modulation of water and electrolyte fluxes in the gut (12), stimulation of oxytocin secretion from isolated rat neurohypophysis (13), regulation of sensory processing (14), and modulation of monoaminergic activity in the medial preoptic area (15). In addition, several lines of evidence also suggest a role for MCH in modification of memory retention (16), modulation of other hormones regulating food intake (17) and sexual behavior (18) and involvement in seizure (19). MCH is also localized in neurons functionally involved in circuits of the extrapyramidal motor systems from striatal centers to the thalamus and cerebral cortex and to the midbrain and spinal cord (20). The complex central nervous system expression pattern of MCH together with the many diverse functions mediated by MCH suggest the existence of more than one MCH receptor.The molecular identities of MCH receptors were unknown until recent reports of the first MCH receptor (MCH-R1) (21-25). MCH-R1 is composed of seven transmembrane domains typical of G protein-coupled receptors and binds to MCH with high affinity. MCH-R1 is coupled to G i , G o , and G q -type G proteins and mediates decreases in intracellular cAMP and increases in intracellular Ca 2ϩ levels, inositol phosphate production, and mitogen-activate protein kinase activity (26). In this report, we describe a novel MCH receptor identified through bioinformatic and molecular cloning approaches. The receptor is similar (35% identical) to the previously published MCH receptor, is able to bind 125 I-labeled MCH, and can be activated by ...

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