ReviewPhylogenetic analysis of the sequences of gastrin-releasing peptide and its receptors: Biological implications
Introduction
Peptides of the bombesin family were first isolated from frog skin. In 1970, Nakajima et al. isolated an undecapeptide, which was named ranatensin, from the skin of the American frog Rana pipiens [1]. A related tetradecapeptide termed bombesin was isolated from the skin of the amphibian Bombina bombina in the same year [2]. A decade elapsed before researchers, using antisera developed against bombesin, identified the mammalian counterpart, which was named gastrin-releasing peptide (GRP) after its first known activity of stimulating gastrin secretion [3]. In 1983, the mammalian homologue of ranatensin was isolated from porcine spinal cord and named neuromedin B (NMB) [4]. The generic name “bombesin-like peptides” is now given to peptides that have significant homology with the carboxyl-terminal sequence of bombesin (Fig. 1).
Cloning of the cDNA encoding human GRP revealed that the peptide is derived from a precursor (preproGRP) of 148 amino acids by cleavage at well-defined sites [5]. Initially the first 23 amino acids of preproGRP are removed by a signal peptidase, and proGRP fragments are therefore numbered from the amino terminal valine. proGRP is then cleaved after the double lysine at positions 29 and 30 by prohormone convertase 2, and a carboxypeptidase B-like activity removes the two basic residues from the carboxyl terminus to form GRP1–28 (glycine-extended GRP1–27, GRPgly). The amidation enzyme peptidyl glycine α-amidating monooxygenase finally removes glyoxylate from glycine 28 and leaves amidated methionine-27 at the C-terminus. The mature form of human GRP contains 27 amino acids, as do porcine [6], chicken [7] and canine GRP [8]. An additional cleavage that ultimately yields GRP18–27 may occur between arginine17 and glycine18 in canine [8], human [9] and porcine [3] GRP, but it is not known whether this cleavage takes place before or after amidation. The sequences of preproGRPs from different species have not been systematically compared.
Four different receptor subtypes (GRP-R [10], [11], NMB-R [11], [12], BRS-3 [13], [14] and BB4 [15]) have now been described for the bombesin-like family of peptides. cDNA clones encoding the GRP receptor (GRP-R) were first isolated from murine Swiss 3T3 cells [10], [16]. Cloning of the human GRP-R from placental and peripheral blood genomic libraries and human small cell lung cancer cell lines revealed 90% homology with the mouse GRP-R [11]. Subsequently the human GRP-R gene was found to contain three exons and was localised to chromosomal band Xp22 [17]. Since binding studies suggested that GRP and NMB exerted their physiological effects through at least two different receptors [18], a GRP-R cDNA probe was used under low stringency hybridization conditions to clone the NMB-R cDNA from a rat oesophageal cDNA library [12]. All the bombesin receptors characterised to date are coupled to guanine nucleotide binding proteins (G-proteins), have seven transmembrane domains, and activate phospholipase C to increase intracellular concentrations of inositol phosphates, diacyl glycerol and calcium [19]. An alternative nomenclature system for the bombesin receptors is also in use [15]. In this system the NMB receptor, GRP receptor and BRS-3 are known as the BB1 receptor, the BB2 receptor and the BB3 receptor, respectively, and the fourth receptor is known as the BB4 receptor.
The bombesin-like peptides play many physiological roles in addition to the stimulation of gastric acid secretion [20], [21]. As well as stimulating the release of a variety of hormones including gastrin, somatostatin and cholecystokinin, the bombesin-like peptides promote exocrine secretion from the pancreas, and smooth muscle contraction in stomach, small intestine, and several other tissues. The neural effects of bombesin-like peptides include changes in animal behaviour, suppression of food intake, and improvement of learning and memory in rodent models. Bombesin-like peptides are also mitogenic for cell lines derived from a number of cancerous tissues [22], [23]. A large body of evidence now suggests that bombesin-like peptides are involved in the development and progression of cancers derived from tissues including lung, prostate, breast, stomach and colon, and may act as autocrine growth factors for various cancers (see [23] for review).
Over the last decade it has become apparent that more than one active peptide may be derived from a single prohormone. Thus although the amidated forms of peptide hormones were thought for many years to be the only biologically active species [24], several glycine-extended hormone precursors such as glycine-extended gastrin and secretin are now known to be active [25], [26]. The glycine-extended form of bombesin is biologically active in various cell lines [27], [28], [29], and acts via the GRP-R [28], [29]. Our own work has also demonstrated that peptides derived from the C-terminal 95 residues of proGRP are bioactive, but that in this case activity is not blocked by GRP-R antagonists [30], [31]. The conservation of sequence noted between mammalian proGRPs in this region was also consistent with a previously unrecognised biological activity [31]. In order to identify conserved and therefore functionally important regions of the proGRP sequence, more extensive species comparisons were undertaken, and are reported in the present paper. At the same time the databases were searched for previously unrecognised members of the GRP receptor family, and the regions conserved between species were identified.
Section snippets
Materials and methods
Sequences were taken from the databases of the National Center for Biotechnology Information (NCBI), the Institute for Genomic Research (TIGR) and the Sanger Institute/European Molecular Biology Laboratory European Bioinformatics Institute (Ensembl). Sequences were aligned with version 1.82 of the program CLUSTALW (http://www.ebi.ac.uk/clustalw/), with the following default settings: gap open penalty, 10; gap extension penalty, 0.2; protein matrix, Gonnet 250. The resultant dendrograms were
Phylogeny of GRP
The sequence of GRP18–27 (Fig. 2A) is well conserved across diverse species ranging from humans to goldfish [5], [6], [7], [8], [9], [33], [34], [35], [36], [37], [38], [39], [40]. After alignment with the program CLUSTALW comparison of the percentage amino acid identity within the proGRP protein sequence between different species as a function of alignment number (Fig. 2B) reveals that the most highly conserved region of the mammalian proGRP sequences is between amino acids 18 and 31. A second
GRP
The amino acid sequence of proGRP shows extensive homology between species (Fig. 2). The region of greatest homology, between alignment numbers 19 and 33, includes the sequence of the known bioactive peptides GRP18–27 and GRP18–28 (glycine-extended GRP18–27). A second region of significant although lower homology is also apparent between alignment numbers 56 and 109.
The observed conservation of amino acids within the C-terminus of proGRP between species (Fig. 2) is consistent with the
Conclusion
In summary it is obvious from the literature that amidated GRP18–27 is a mitogen for many cancers including lung, prostate and breast carcinoma. However, the situation with gastrointestinal cancer is less clear with active debate over the presence and nature of GRP, the biological activity of GRP, the frequency and subtype of GRP receptors, and the mechanisms of their action [23]. The observation described here that the C-terminal extension of the proGRP sequence is highly conserved over a wide
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2020, International Journal of Biological MacromoleculesCitation Excerpt :The amphioxus GRPs were found to locate at the basal position of three different clusters (Fig. 2A), suggesting that the bombesin peptide family in vertebrates may originate from a common ancestral gene, and the grp gene in amphioxus might represent the ancestral type. Four different subtypes of bombesin receptor members have been identified in vertebrates [7]. However, only one grp receptor (grpr) gene was found by searching the genome of amphioxus B. belcheri.
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2018, PeptidesCitation Excerpt :Subsequently, rat and human GRP receptors were cloned [15], and chicken GRPR was cloned by reverse transcription-PCR [16]. Sequence analysis of the receptors of different species showed that GRPR belongs to the 7-membrane spanning domains superfamily; the most highly conserved regions occurred in transmembrane helices 2, 3, 5–7 and in the third intracellular loop [17]. Previous studies using RT-PCR, in situ hybridization, binding studies, immunohistochemical studies and so on have shown that GRPR is mainly distributed in the CNS and peripheral tissues of many animals.
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2015, PeptidesCitation Excerpt :In this study [355] no correlation was found between the affinities of the different BnR agonists for rat and human GRPRs (r = 0.313, p = 0.54). The in vivo synthesis of GRP and NMB involves their cleavage from a pre-proform (PreproGRP-148 amino acids, preproNMB-76 amino acids) which consist of a signal peptide, the hormone sequence and a carboxyl terminal extended peptide [20,245,331]. The COOH terminal extended forms of GRP/Bn have low affinity for the GRPR (IC50 >1 μM), however, recently a number of papers have reported biologically active effects of COOH terminal extended Bn/GRP analogs [20,122,257].
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