Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

Abstract

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC 50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and͞or GPR8.T here are a large number of orphan G protein-coupled receptors (GPCR) whose cognate ligands have yet to be identified (1, 2). The search for endogenous ligands of orphan GPCRs is important because GPCRs are in general excellent drug targets (3). GPR7 and GPR8 are two orphan GPCRs (4). They were originally cloned from human genomic DNA by degenerative PCR using primers based on the sequences of the ␦-opioid receptor and somatostatin receptor. Indeed, GPR7 and GPR8 each share Ϸ40% overall amino acid identities with opioid and somatostatin receptors. Human GPR7 and GPR8 are 70% identical to each other at the nucleotide level and 64% identical at the amino acid level. Interestingly, no orthologue exists for the GPR8 gene in rodents, indicating that GPR8 may have originated as a replicate of GPR7 after divergence of the rodent from other species in mammalian evolution (5).The observation that GPR7 and GPR8 mRNAs are expressed in distinct areas in the central nervous system (CNS) (4, 5), together with the similarity of GPR7 and GPR8 with opioid and somatostatin receptors, strongly argue for the existence of endogenous peptide ligand(s) in CNS. In this study, we have purified a neuropeptide ligand for GPR7 from the bovine hypothalamus. This peptide, termed neuropeptide B (NPB), consists of 29 aa with a unique brominated N-terminal tryptophan. Through EST database searches, we also identified another isopeptide of the same family, termed neuropeptide W (NPW). While this manuscript was being prepared, three papers were published reporting two endogenous ligands for GPR7 and GPR8 (6-8). Sequ...

Neuropeptide W exerts a potent suppressive effect on blood leptin and insulin concentrations in the rat

“…This rather unexpected finding suggests that the primary effect of NPW on leptin secretion is to lower its level in general circulation. This effect may explain the findings of Tanaka et al (8) demonstrating NPB-induced hyperphagia within the first 2 h of the experiment, followed by hypophagia. On the other hand, Ishii et al (7) suggest that obesity in NPBWR1 -/-mice is independent of leptin and melanocortin signaling.…”

“…As known, both neuropeptides and their receptors are structurally highly homologous and this suggests that each ligand and receptor are partially overlapping and also have specific roles in this signaling system. As demonstrated, NPB activates and binds to both human NPBWR1 (with higher affinity) and NPBWR2, and similar effects are demonstrated with NPW23 and NPW30, with NPBWR1 binding with lower affinity (6,8). Previous observations revealed notable differences in NPB and NPW action on both rat and human adrenocortical cells (18,20,24).…”

“…Intracerebroventricular infusion of NPB in mice induced hyperphagia during the first 2 h, followed by hypophagia (8). On the other hand, male NPBWR1 -/-mice developed adultonset obesity (7).…”

“…The amino acid sequence of NPW23 is identical to the first 23 Nterminal amino acids of NPW30 (4,5). Expression of NPB, NPW and their receptor gene was found to be present in the central nervous system and in the peripheral organs of humans, rats, and mice (1,2,(5)(6)(7)(8)(9). Distribution of the gene expression within the brain suggests that NPB plays a role in the control of feeding and neuroendocrine axis functions, as well as in memory and learning processes.…”

“…NPW in turn, affects feeding, stimulates prolactin secretion, and regulates the function of the hypothalamic-pituitary-adrenal axis. Intracerebroventricular infusion of NPB or NPW affects food intake and energy expenditure (5,8,(10)(11)(12). Furthermore, targeted disruption of the NPBWR1 gene leads to metabolic defects and adult-onset obesity (7).…”