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Neuropeptide B (NPB) and Neuropeptide W (NPW)

Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8.

Four peptides, L7, L7C, L8 and L8C, have been identified as natural ligands for orphan GPCR GPR7 and GPR8. The sequence of L7 and L8 is also identical to NPB and NPW:

L7 (Human) = NPB-23, des-Br (Human);
L7C (Human) = NPB-29, des-Br (Human);
L8 (Human) = NPW-23 (Human);
L8C (Human) = NPW-30 (Human).

Brezillon et al. J Biol Chem. 2003 Jan 10;278(2):776-83.

Targeted disruption of GPR7, the endogenous receptor for neuropeptides B and W, leads to metabolic defects and adult-onset obesity.

Gold-thioglucose (GTG) induces lesions in the ventromedial nucleus of the hypothalamus, resulting in hyperphagia and obesity. To identify genes involved in the hypothalamic regulation of energy homeostasis, we used a screen for genes that are dysregulated in GTG-induced obese mice. We found that GPR7, the endogenous G protein-coupled receptor for the recently identified ligands neuropeptide B and neuropeptide W, was down-regulated in hypothalamus after GTG treatment. Here we show that male GPR7-/- mice develop an adult-onset obese phenotype that progressively worsens with age and was greatly exacerbated when animals are fed a high-fat diet. GPR7-/- male mice were hyperphagic and had decreased energy expenditure and locomotor activity. Plasma levels of glucose, leptin, and insulin were also elevated in these mice. GPR7-/- male mice had decreased hypothalamic neuropeptide Y RNA levels and increased proopiomelanocortin RNA levels, a set of effects opposite to those evident in ob/ob mice. Furthermore, ob/ob GPR7-/- and Ay/a GPR7-/- double mutant male mice had an increased body weight compared with normal ob/ob or Ay/a male mice, suggesting that the obesity of GPR7-/- mice is independent of leptin and melanocortin signaling. Female mice did not show any significant weight increase or associated metabolic defects. These data suggest a potential role for GPR7 and its endogenous ligands, neuropeptide B and neuropeptide W, in regulating energy homeostasis independent of leptin and melanocortin signaling in a sexually dimorphic manner.
Ishii et al. Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10540-5.

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

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.
Tanaka et al. Proc Natl Acad Sci U S A. 2003 May 13;100(10):6251-6.

Identification of a neuropeptide modified with Bromine as an endogenous ligand for GPR7

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 (O'Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995) Genomics 28, 84-91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O'Dowd, B. F. (1999) Mol. Brain Res. 71, 96-103). 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.
Fujii et al. J Biol Chem. 2002 Sep 13;277(37):34010-6. Epub 2002 Jul 12.

Identification of neuropeptide W (NPW) as the endogenous ligand for orphan G-protein-coupled receptors, GPR7 and GPR8

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.
Shimomura et al. J Biol Chem. 2002 Sep 27;277(39):35826-32. Epub 2002 Jul 18.

Mapping in Rat Brain & Hypothalamus (H-005-56)

  

Tissue Sample

Rat Brain & Hypothalamus

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

Intact

Blocking

2% Normal Goat Serum

Primary Antibody

Rabbit Anti-NPB-29 (Rat) Serum (Catalog No.:H-005-56)

Optimal Dilution

1:200~500 (1hour at RT) 

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

ABC (Vector) (1:400, 30 min)

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

Mapping in Rat/Mouse Hypothalamus & Rat Brain(H-005-61)

Tissue Sample

Rat & Mouse Hypothalamus; Rat Brain Tissue

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

Target Retrieval 25 min (Steam)

Blocking

2% Normal Goat Serum

Primary Antibody

Rabbit Anti-Neuropeptide W-23 (NPW-23) (Mouse, Rat) Serum (Catalog No.: H-005-61)

Optimal Dilution

1:100 (Mouse Hypothalamus) , 1:200 (Rat Hypothalamus) (1hour at RT)

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

ABC (Vector) (1:400, 30 min)

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

Neuropeptide B immunoreactivity in the central nervous system of the rat

Siok L. Dun a, G. Cristina Brailoiua, Keisuke Mizuoa, Jun Yangb, Jaw Kang Changa, Nae J. Duna,*
aDept. of Pharmacology, Temple Univ. School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, US;
bPhoenix Pharmaceuticals, Inc., Belmont, CA 94002, USA

Confocal images of rat midbrain (A– C) and hypothalamic sections (D–F) double labeled with neuropeptide B antiserum (NPB, green image) and tyrosine hydroxylase (TH) or vasopressin (VP) antibody. In the substantia nigra pars compacta, all the irNPB cells (A) are TH positive (B). (C) A merged image of panels A and B. In the supraoptic nucleus, some of the VP-neurons (E) express NPB immunoreactivity (D). (F) A merged image of panels D and E, where co-expression of NPB and VP immunoreactivity is clearly detected in several neurons. Scale bar: 20 µm.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Photomicrographs of sections through the rat hypothalamus labeled with or without NPB antiserum. (A) Strongly labeled neurons are seen in the lateromagnocellular part (PaLM) and medial parvocellular part (PaMP) of the paraventricular hypothalamic nucleus and in the supraoptic nucleus (SO). Scattered cells are present in the periventricular hypothalamic nucleus (Pe) and retrochiasmatic nucleus (RCh). (B) A higher magnification of the top area outlined in panel A where many immunoreactive cells from PaLM and PaMP are clearly visible; smaller, positively labeled cells are seen in the Pe. (C) A higher magnification of the bottom area outlined in panel A, where labeled cells are present in SO. (D) A hypothalamic section processed without NPB antiserum; immunoreactivity is not noted in this section. Abbreviations: f, fornix; opt, optic tract; 3V, 3rd ventricle. Scale bar: panels A and D, 250 µm; panels B and C, 100 µm.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Photomicrographs of sections through the caudal rat hypothalamus labeled with NPB antiserum. (A) Intensely labeled cells are seen in the supraoptic retrochiasmatic nucleus (SOR); immunoreactive cells were also present in zona incerta (ZI), perifornical area, lateral hypothalamic area (LH), dorsal hypothalamic area (DA), dorsomedial hypothalamic nucleus dorsal part (DMD), and tuber cinereum area (TC). (B) A higher magnification of the top area outlined in panel A, where moderately labeled irNPB neurons are present in the zona incerta (ZI). (C) A higher magnification of the area around the fornix (f) in panel A, where immunoreactive cells are seen surrounding the fornix. (D) A higher magnification of the bottom area outlined in panel Awhere strongly labeled neurons are present in SOR. Abbreviations: 3V, 3rd ventricle. Scale bar: panel A, 250 µm; panels B– D, 50 µm.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Photomicrographs of sections through the rat hypothalamus and pituitary labeled with NPB antiserum. (A) Intensely labeled fibers are present in the internal layer of the median eminence (MEI) and few small cells are present in the arcuate nucleus (Arc; arrows). (B) A section of posterior pituitary where NPB-immunoreactive cell processes/terminals and occasionally small cells are noted. (C) Intensely labeled fibers are noted in the external layer of the median eminence (MEE). (D) A section of anterior pituitary where many small NPB-labeled cells are seen; arrows point to representative cells. Abbreviations: 3V, 3rd ventricle. Scale bar: panels A and C, 250 µm; panels B and D, 100 µm.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Photomicrographs of sections through the rat midbrain labeled with NPB antiserum. (A) Immunoreactive cells are present in Edinger–Westphal nucleus (EW), substantia nigra: compact part, dorsal tier (SNCD), reticular part (SNR), lateral part (SNL) and medial part (SNM), paranigral nucleus (PN), ventral tegmental area (VTA), and interfascicular nucleus (IF). (B) A higher magnification of the area outlined in panel A showing many immunoreactive neurons within the substantia nigra; there is a higher density of cells in the SNM and SNCD as compared to the SNR. (C) A higher magnification of the area outlined in panel Awhere irNPB neurons are seen in the IF, PN, and VTA. (D) Intensely labeled neurons are present in the EW. Abbreviations: IP, interpeduncular nucleus. Scale bar: panel A, 250 µm; panels B– D, 100 µm.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Immunohistochemistry

Rats anesthetized with urethane (1.2 g/kg ip) were intracardially perfused with chilled 0.1 M phosphatebuffered saline (PBS) followed by freshly prepared 0.2% picric acid/4% paraformaldehyde in PBS. Brains and spinal cords were removed, postfixed in the same fixative for 2 h, and immersed in 30% sucrose/PBS solution overnight. Coronal brain or transverse spinal cord sections of 40 µm thick were prepared with the use of a Vibratome and processed for NPB immunoreactivity (irNPB) by means of the standard avidin–biotin complex procedures. Tissues were first treated with 3% H2O2 to quench endogenous peroxidase, washed several times, and blocked with 10% normal goat serum. Tissues were then incubated in NPB antiserum for 48 h at 4 -C with gentle agitation. The NPB antiserum, a rabbit polyclonal (Phoenix Pharmaceuticals, Inc., Belmont, CA), was directed against the rat NPB. The antiserum was used at a dilution of 1:1000 with 0.4% Triton X-100 and 1% bovine serum albumin in PBS. After thorough rinsing, sections were incubated in biotinylated anti-rabbit IgG (1:150, Vector Laboratories, Burlingame, CA) for 2 h. Sections were rinsed with PBS and incubated in avidin–biotin complex solution for 1 h (1:100, Vector Laboratories). After several rinses in Tris-buffered saline, sections were developed in diaminobenzidine/H2O2 solution and washed for at least 2 h with Tris-buffered saline. Sections were mounted on slides with 0.25% gel alcohol, air-dried, dehydrated with absolute alcohol followed by xylene, and coverslipped with Permount.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

Double labeling procedures

In the case of double labeling, the technique of sequential labeling with primary antibodies from different species was used. Tissues were first blocked with 10% normal goat serum and then incubated in NPB antiserum (1:500 dilution with 0.4% Triton X-100 and 1% bovine serum albumin in PBS) for 48 h in a cold room with gentle agitation. Following several washes with PBS, sections were incubated in biotinylated anti-rabbit IgG (1:50, Vector Laboratories) for 2 h. Subsequent to several washes in PBS, tissues were incubated for 4 h in Fluorescein Avidin D (1:50, Vector Laboratories). After thorough rinsing with PBS for 2 h, tissues were blocked with normal donkey serum and incubated in either tyrosine hydroxylase monoclonal antibody, monoclonal oxytocin antibody, or vasopressin antiserum, a guinea pig polyclonal , for 48 h in a cold room with gentle agitation. After washing with PBS for 30 min, tissues were incubated with mouse IgG Texas red or guinea pig IgG Texas red for 4 h (1:50, Jackson Laboratories). Lastly, tissues were washed for 30 min with PBS, mounted in Citifluor (Ted Pella, Redding, CA), and coverslipped. Sections were examined under a confocal scanning laser microscope (Leica TCS SL) with excitation/emission wavelengths set to 488/520 nm for FITC and 543/620 nm for Texas red in the sequential mode.
Dun et al. Brain Res. 2005 May 31;1045(1-2):157-63. Epub 2005 Apr 14

NPB-29 (Human) - EIA Kit (EK-005-51)

NPB-29 (Rat) - EIA Kit (EK-005-56)

NPB-29 (Human) - RIA Kit (RK-005-51)

NPB-29 (Mouse) - RIA Kit (RK-005-58)

NPW-23 (Human) - RIA Kit (RK-005-60)

NPW-23 (Rat, Mouse) - RIA Kit (RK-005-61)

Normal Plasma Levels of Neuropeptide B-29 (NPB-29) in healthy normal subjects
Using Phoenix Pharmaceuticals' NPB-29 RIA Kit (Cat No: RK-005-51)
Non-extracted samples
Extracted samples
201.45± 10.2 pg/ml*
26.7± 5.4 pg/ml#
Normal Plasma Levels of Neuropeptide W-23 (NPW-23) in Sprague Dawley rats
Using Phoenix Pharmaceuticals' NPW-23 RIA Kit (Cat No: RK-005-61)


Non-extracted samples
Extracted samples
97.6 ± 14.7 pg/ml*
30.7 ± 3.0 pg/ml#


Data are represented as mean ± SEM, n=7
.
* 200 ul of each plasma sample was lyophilized and dissolved with 100 ul of RIA buffer to perform the assay.
# Plasma samples were extracted using Phoenix Pharmaceuticals' C18 plasma extraction protocol.

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.
Tanaka et al. Proc Natl Acad Sci U S A. 2003 May 13;100(10):6251-6.

Prepro-Neuropeptide B (Human) Prohormone Schematic

 

Comparison of Prepro-Neuropeptide B Amino Acid Sequence between Human, Rat, Mouse & Bovine.

Human Prepro-Neuropeptide W (NPW) Prohormone Schamtic

Amino acid Sequence of Human Prepro-Neuropeptide W (NPW)

Comparison of Neuropeptide W-30 (NPW-30) and DesBr-Neuropeptide B-29 (DesBr-NPB29)

Comparison of amino acid sequence of prepro-L7 and prepro-L8

Functional activity of Phoenix NPB on human GPR7 receptor

Dr. Hans-Peter Nothacker, Dept. of Pharmacology, UC Irvine

In vivo pharmacological effects of i.c.v.-injected NPB on food intake

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).
Tanaka et al. Proc Natl Acad Sci U S A. 2003 May 13;100(10):6251-6.

 

Interaction of NPB with GPR7 and GPR8
 
Inhibition of cAMP Production IC50 (nM)
Binding* IC50 (nM)
   
Bovine
Human

 Human

  Peptide
GPR7
GPR8
GPR7
GPR8
GPR7
Bovine NPB-29
1.9
4.0
0.58
9.7
0.34
  DesBr-NPB-29**
1.2
5.4
0.43
8.1
0.31
Human NPB-29
1.8
51
0.44
32
0.32
  DesBr-NPB-29
2.6
52
0.45
49
0.33
  DesBr-NPB-23
3.5
47
0.58
44
1.6
  NPW-23
2.4
4.8
0.82
3.7
0.40
Rat DesBr-NPB-29
1.2
14
0.86
8.8
0.34
  *Binding potency was calculated by the competition of peptides in the binding of [125I-Tyr]-desBr-NPB-23 to human GPR7 ;
**Des-Br-NPB indicates NPB without bromination

 
Functional activity and binding affinity of human NPW for GPR7 and GPR8
 
 
Human GPR7
Human GPR8
 
cAMP
Binding
cAMP
Binding
 
IC50 (nM)
IC50 (nM)
Human NPW-23
0.025±0.004
0.096±0.007
0.178±0.007
0.210±0.021
Human NPW-30
0.133±0.034
0.025±0.005
1.244±0.131
0.021±0.002
 
Functional activity and binding affinity
 
 
Human GPR7
Human GPR8
 
Kd (pM)
Bmax (pmol/mg)
Kd (pM)
Bmax (pmol/mg)
 
125I-NPW-23 (H)
31.8±3.0
2.02±0.10
20.7±0.6
4.37±0.04
125I-Tyr11-desBr-NPB-23(H)
36
1.3

%005-49%;%005-51%;%005-52%;%005-53%;%005-54%;%005-55%;%005-56%;%005-57%;%005-58%;%005-59%;%005-66%;%005-67%;%005-68%;%005-69%;%005-70%;%005-60%;%005-61%;%005-62%;%005-63%;%005-64%;%005-65%;%005-73%;%005-74%;%005-75%;%005-76%


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