Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy

Abstract

The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Binding experiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confirmed the bombesin-like nature of the cloned receptor.The relative order of ligand affinity, GRP = neuromedin C & neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.Two related peptides, ranatensin and bombesin, found in frog skin (Nakajima et al., 1970;Anastasi et al., 1971) were the first members of a large family of regulatory peptides, named later bombesin-like peptides. To date, more than 20 peptides of this family are known to share a common amino acid sequence at their C-terminus. The peptides can be classified into three subfamilies according to their C-terminal tripeptide; the bombesin, the ranatensin/litorin and the phyllolitorin subfamilies (Erspamer, 1988). To date, there are only three bombesin-like peptides identified in mammalian tissues; gastrin-releasing peptide (McDonald et al., 1979), neuromedin B (Minamino et al., 1983) and neuromedin C (Minamino et al., 1984). Gastrin-releasing peptide (GRP) and neuromedin C (C-terminal decapeptide of GRP) belong to the bombesin subfamily and neuromedin B belongs to the ranatensin/litorin subfamily (Fig.

Autocrine and paracrine signaling through neuropeptide receptors in human cancer

“…Seven membrane-spanning receptors exhibit-ing selectivity for GRP or NMB have been identi®ed through molecular cloning approaches (reviewed in Battey and Wada, 1991;Ohki-Hamazaki, 2000). In addition, a distinct receptor referred to as bombesin receptor subtype-3 (BRS-3) with extensive homology to the GRP and NMB receptors has been cloned, for which the endogenous ligand remains unde®ned (Fathi et al, 1993;Gorbulev et al, 1992). Interestingly, mice in which the BRS-3 gene has been disrupted develop multiple metabolic defects and mild obesity, indicating that BRS-3 and its unde®ned ligand also regulate important endocrine and metabolic processes (OhkiHamazaki et al, 1997).…”

Bombesin, obesity, and social behavior

“…2 In addition, a third subtype of mammalian bombesin receptors (BRS-3) has been cloned: however, high affinity natural ligand(s) specific to BRS-3 have not been identified yet. 3,4 Mammalian bombesin-like peptides and their receptors are widely distributed in the central nervous system and peripheral organs. They modulate exocrine and endocrine processes, smooth muscle contraction, metabolism, homeostasis, and behavior.…”

Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus

“…To localize the receptor gene on a chromosome we carried out the PCR analysis on chromosomal DNA from 14 human-rodent somatic cell hybrids [l l] using two HSUBR-specific primers (see Section 2). The primers were designed to the C-terminal part of the receptor and to the 3' non-coding region, to regions where there is the highest difference between HSUBR and two other human bombesin receptor genes [7], from one side, and also between HSUBR and PU18 [8], from the other side. In control PCR experiments with chromosomal DNA from human cell lines a single 349 bp fragment was observed.…”

“…3 min. PCR products were resolved on agarose gel, transferred to Hybond-N and hybridized to the 3ZP-labelled 1.5-kb EcoRI-PstI fragment of PU61 clone [8]. Amplified DNA was cloned mto pGEM-7Zf(+) (Promega) and partially sequenced.…”

“…Both types of human and rodent receptors have been cloned and characterized [5- cloned a new bombesin receptor subtype from guinea pig that is expressed in uterus during pregnancy [8]. The similarity between this receptor and the two other cloned rodent bombesin receptors, the GRP receptor and the neuromedin B receptor, is 52% and 47%, respectively, i.e.…”