Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 100 (10), 6251-6

Characterization of a Family of Endogenous Neuropeptide Ligands for the G Protein-Coupled Receptors GPR7 and GPR8

Affiliations

Characterization of a Family of Endogenous Neuropeptide Ligands for the G Protein-Coupled Receptors GPR7 and GPR8

Hirokazu Tanaka et al. Proc Natl Acad Sci U S A.

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 (EC(50)) 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 andor GPR8.

Figures

Figure 1
Figure 1
Amino acid sequences of NPB and NPW. (A) Amino acid sequences of mature NPB and NPW peptides. An asterisk indicates the posttranslational bromination site of the native bovine NPB. Peptide sequences of other species are deduced from cDNA sequences. Amino acid identities between NPB and NPW are shown in black. Shaded residues are conserved only within the NPB or NPW. (B) Deduced amino acid sequences of prepro-NPB and NPW precursor polypeptides. Mature peptides are marked by equal signs. Question marks indicate undetermined sequence of bovine prepro-NPB. Human and mouse prepro-NPW cDNA do not have a translation initiator ATG codon; putative translation initiation sites are indicated by a pound sign. An arrow indicates a possible additional processing site for NPW. Identical amino acids within the orthologues are shown in black. Lightly shaded residues are conserved in more than four of six (NPB) or two of three (NPW) species.
Figure 2
Figure 2
In vitro pharmacology of NPB and NPW assessed by melanophore pigment aggregation assay (A and B) and [Ca2+]i transient assay (C) in mammalian cells. (A and B) X. laevis melanophores were transiently transfected with human GPR7 (A) or GPR8 (B) cDNA, and challenged with various forms of synthetic human NPB and NPW peptides. Brominated NPB and nonbrominated NPB have approximately equal potencies on GPR7 (A), whereas both of them have much weaker potencies on GPR8 (B). Brominated NPW and nonbrominated NPW both have similar potencies on GPR7 and GPR8. des[l-Trp-1]NPB and des[l-Trp-1]NPW are both much less potent on GPR7 and GPR8. Data are the mean of eight separate experiments. (C) Ltk− cells were stably cotransfected with the human GPR7 cDNA and Gqi chimera cDNA, and challenged with nonbrominated synthetic human NPB and NPW. NPB has a modestly higher potency than NPW. When Gqi cDNA is not cotransfected, neither NPB nor NPW could induce the calcium mobilization (open and filled circles are all overlapping). Peak [Ca2+]i values are represented as percentages of the maximum response. Data are presented as the mean ± SEM of three experiments performed in duplicate.
Figure 3
Figure 3
In situ hybridization of NPB (A–H) and NPW (I–L) mRNA on mouse brain sections. (Scale bars are 300 μm.) (AH) NPB mRNA in mouse brain. CA1–3, CA1–3 field of hippocampus; LHb, lateral habenular nucleus; PaMP, paraventricular hypothalamic nucleus medial parvicellular part; EW, EW nucleus; m5, motor root of the trigeminal nerve; s5, sensory root of the trigeminal nerve; LPBI, lateral parabrachial nucleus internal part; Me5, mesencephalic trigeminal nucleus; Sub CA, subcoeruleus nucleus α part; A5, noradrenergic cell group A5; LC, locus coeruleus; IOB, inferior olive subnucleus B. (IL) NPW mRNA in mouse brain. PAG, periaqueductal gray matter; EW, EW nucleus; VTA, ventral tegmental area; DR, dorsal raphe nucleus; DRD, dorsal raphe nucleus dorsal part.
Figure 4
Figure 4
In vivo pharmacological effects of i.c.v.-injected NPB on food intake (A and B), locomotor activity (C), and nociception (D and E). (A) Vehicle or 3 or 10 nmol of synthetic rat NPB was i.c.v. injected in bolus into freely fed mice, and food consumption was measured. Injections were performed at 20:00 (beginning of dark phase) and food intake was measured at 22:00, 00:00, and 08:00 the next morning (end of dark phase). The data represent food intake between the indicated times (mean ± SEM, n = 4–5 per group). Asterisks indicate significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (B) Anorexic effect of NPB is enhanced by pretreatment with CRF. CRF (0.3 nmol) was i.c.v. injected 15 min before the injection of 3 nmol of synthetic rat NPB in mice. NPB was i.c.v. injected at 20:00 (beginning of dark phase), and food intake was measured at 22:00 and 00:00. n.d., no food intake detected (bars invisible). The data represent food intake between designated times in dark phase (mean ± SEM, n = 7 per group). Asterisks indicate the significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (C) NPB-induced hyperlocomotion in both dark and light phases. Three nanomols of synthetic rat NPB or vehicle was i.c.v. injected into rats placed in an open field apparatus. The data represent the distance traveled (meters) per 2 h (mean ± SEM, n = 6–8 per group). Asterisks indicate significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (D) Paw flick tests were performed 20 min after i.c.v. injection of vehicle or 3 nmol of synthetic rat NPB in rats. Tests were performed without (Car−) and with (Car+) the chemical inflammation induced by carrageenan. Two milligrams of carrageenan was injected into a hindpaw 3 h before the paw flick test. The data represent the latency (seconds) to flick the paw out of the path of the heat-producing light beam (mean ± SEM, n = 6–8 per Car− group and n = 4–5 per Car+ group). (E) Formalin tests were performed 10 min after injection of vehicle, 3 nmol of nonbrominated synthetic rat NPB, or 3 nmol of brominated NPB in rats. The data represent the licking duration (seconds per 5-min bin) at indicated minutes after injection of 50 μl of 5% formalin into a hindpaw (mean ± SEM, n = 4–5 per group). Asterisks indicate significant difference from vehicle treatment (P < 0.05, one-way ANOVA, Fisher's post hoc analysis).

Similar articles

See all similar articles

Cited by 41 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback