The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system. Searches for ligands for orphan G-protein-coupled receptors (GPCRs)1 have discovered many novel peptides and have identified the previously unknown receptors of bioactive substances (1-9). Studies on the newly identified ligands and their receptors have given us a more precise understanding of the physiological processes involved in the endocrine, cardiovascular, reproductive, immune, inflammatory, digestive, metabolic, and central nervous systems (1-11). In addition, these studies have provided opportunities to discover innovative drugs that exert their pharmacological effects by interacting with an identified receptor as an agonist or antagonist (12). GPR7 and GPR8, for which the ligands have not been identified, are structurally related orphan GPCRs. Two genes for GPR7 and GPR8 were originally isolated from human genomic DNA by O'Dowd et al. (13). Human GPR7 highly resembles human GPR8, with an amino acid identity of 64%. Among various families of GPCRs, GPR7 and GPR8 share high similarity to the opioid and somatostatin receptor families. In mammalian brain, gene expression of GPR7 and GPR8 was detected by Northern blot and in situ hybridization analyses (13). Especially in rat brain, GPR7 mRNA was detected in regions of the cortex, hippocampus, and hypothalamus (14). Profiles of GPR7 and GPR8 expressed mainly in brain suggest that the endogenous ligands for the two receptors have several functions in the central nervous system.In this study, we report the purification, cloning, and characterization of neuropeptide W (NPW). We attempted to purify the agonist peptide for GPR8. The cDNA encoding the agonist peptide for GPR8 demonstrates the existence of neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30), which exhibit no meaningful similarity to any known peptides. With the functional and binding characterization of NPW for GPR7 and GPR8, we show that NPW is the endogenous ligand for both of these recept...

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 ( The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. niJDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeatedsequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.

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