Research reportCloning and expression of the neuromedin B receptor and the third subtype of bombesin receptor genes 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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2017, Molecular MetabolismCitation Excerpt :Mammalian BRS-3 may be conserved during evolution because it can modify the response pattern of other GPCRs or contribute to basal signaling [11]. Brs3 mRNA and BRS-3 binding activity are located in restricted regions of the brain, including portions of the hypothalamus, amygdala, and thalamus [12–19]. BRS-3 is also reported to be present at low levels in peripheral sites including pancreatic islets, developing testis, female reproductive tract, lung, and muscle, and in certain cancers [5,20–22] (https://gtexportal.org/home/gene/BRS3).
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2014, Neurobiology of Learning and MemoryCitation Excerpt :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.