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...