Cloning and expression of the neuromedin B receptor and the third subtype of bombesin receptor genes in the mouse

doi:10.1016/S0006-8993(97)00380-6

Brain Research

Volume 762, Issues 1–2, 11 July 1997, Pages 165-172

https://doi.org/10.1016/S0006-8993(97)00380-6 Get rights and content

Abstract

We cloned the genes for the mouse homologue of the neuromedin B receptor (NMB-R) and the bombesin receptor subtype 3 (BRS-3). Both receptor genes consist of three exons with well-conserved intron-exon borders. Although the NMB-R gene spans more than 10 kb, the BRS-3 gene spans only about 4 kb. Comparison of the mouse and human receptor sequences indicates 90% (NMB-R) and 85% (BRS-3) sequence homology at the amino-acid level. In the adult mouse, the NMB-R mRNA is expressed in the brain, testis, esophagus, intestine and uterus, whereas the BRS-3 mRNA is expressed predominantly in the brain. In the brain, the NMB-R gene expression is prominent in the thalamic and olfactory regions, and the BRS-3 gene is expressed particularly in the hypothalamic region. In mouse testis, the NMB-R gene expression is prominent, and the expression of BRS-3 mRNA is barely detected. In contrast, BRS-3 has been shown to be expressed in rat testis and guinea-pig uterus, therefore it is possible that a different subtype of the bombesin receptor mediates the same response in different species. Together with the mouse GRP-R gene cloned previously, cloning of the mouse NMB-R and BRS-3 genes permits comparison of function and structure of the three bombesin receptor subtypes in the mouse.

Introduction

Bombesin is a tetradecapeptide originally isolated from amphibians [1]. Two mammalian peptides related to bombesin, gastrin-releasing peptide (GRP) [14]and neuromedin B (NMB) [15]have been isolated and characterized. Mammalian bombesin-like peptides are widely distributed in both the central nervous system and digestive tracts, and play an important role in modulating behavior, metabolism and exocrine as well as endocrine processes [for review, see [13]]. These processes include endocrine and exocrine secretion by the gastrointestinal tract, pituitary secretion, gastrointestinal motility, thermoregulation, food intake, and grooming behavior. They also function as growth factors for some cell lines. Their biological functions are mediated by binding to high-affinity receptors on the cell surface. Mouse, rat and human cDNA clones encoding a GRP preferring receptor (GRP-R) as well as rat and human cDNA clones encoding a NMB preferring receptor (NMB-R) have been isolated and characterized 2, 4, 18, 20. Additionally, a third subtype of bombesin receptor (BRS-3) which shows low affinity for both GRP and NMB has been cloned in the human and guinea-pig 7, 8, 9.

To elucidate a molecular basis of varied functions of bombesin-like peptides in mammals, we compared the characteristics of three bombesin receptors in the same species. For this purpose, we isolated and characterized mouse NMB-R and mouse BRS-3 genes. In this paper, we report the genomic structure, amino-acid sequence and mRNA tissue distribution of both genes in the mouse, enabling comparison of the characteristics of GRP-R, NMB-R and BRS-3 in mouse.

Section snippets

Isolation of the mouse NMB-R and BRS-3 genomic clones

Approximately 1×10⁶ plaques from the 129SV mouse genomic library (Stratagene) were screened with ³²P-labeled rat NMB-R [20]and human BRS-3 [7]cDNA probes. Filters were hybridized overnight at 65°C in buffer (4×SSC, 5×Denhardt's solution, 0.5% SDS and 2 μg/ml herring sperm DNA) containing 8×10⁵ cpm/ml denatured probe. Filters were washed 3× at 55°C for 20 min in 2×SSC, 0.1% SDS. Two positive plaques of mouse NMB-R candidates and five positive plaques of mouse BRS-3 candidates were

Results

Mouse 129SV genomic library was screened at high stringency with a rat NMB-R cDNA probe or with a human BRS-3 cDNA probe. Two candidate clones of NMB-R gene and five candidate clones of BRS-3 gene were isolated and subcloned into plasmids. Restriction mapping and Southern hybridization revealed that two candidate clones of NMB-R gene were derived from almost the same genomic region, although one clone contained a larger insert than the other. Similarly, the five candidate clones of the BRS-3

Discussion

It has been known that bombesin and its related peptides elicit a wide spectrum of biological activity by binding to high-affinity receptors on the cell surface. In this report, we describe the genomic structure and pattern of expression of two types of mouse bombesin receptors, NMB-R and BRS-3.

The predicted proteins deduced from the mouse NMB-R and BRS-3 genes are 390 and 399 amino acids long, respectively. Coding regions are separated by two introns in both genes. The position of the first

Acknowledgements

This investigation was supported in part by research grants from the Ministry of Education, Science, Sports and Culture, the Ministry of Health and Welfare, the Science and Technology Agency of Japan, the Japan Health Science Foundation and the Japan Foundation for Neuroscience and Mental Health.

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GRPR binds preferentially to GRP, with lower affinity for another mammalian bombesin-like peptide, neuromedin B (NMB) (Jensen & Gardner, 1981; Ladenheim, Jensen, Mantey, McHugh, & Moran, 1990; Moody, Getz, O’Donohue, & Rosenstein, 1988; Moody, Staley, Zia, Coy, & Jensen, 1992; von Schrenck et al., 1990; Wang et al., 1992). The other types of bombesin receptors described in mammals are the NMB receptor (NMBR), which has higher affinity for NMB than GRP (Wada et al., 1991), and BRS-3, which shows low affinity for all known bombesin-like peptides and is considered an orphan receptor (Fathi et al., 1993; Gorbulev, Akhundova, Buchner, & Fahrenholz, 1992; Jensen et al., 2008; Ohki-Hamazaki, Wada, Matsui, & Wada, 1997; Whitley, Moore, Giraud, & Shulkes, 1999). The current terminology adopted by several receptor classification guides uses the names BB1, BB2, and BB3 for NMBRs, GRPRs, and BRS-3 receptors respectively.
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Research reportCloning and expression of the neuromedin B receptor and the third subtype of bombesin receptor genes in the mouse

Abstract

Introduction

Section snippets

Isolation of the mouse NMB-R and BRS-3 genomic clones

Results

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

Neuron

Research report
Cloning and expression of the neuromedin B receptor and the third subtype of bombesin receptor genes in the mouse