Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the ‘gut-brain peptide’ systems.

HS) 32 33 ¶ These authors contributed equally to this work. 62 Author Summary 63 64Bombesin is a putative antibacterial peptide isolated from the skin of the frog, 65Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and 66 neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery 67 has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and 68 their receptors in vertebrates. We have classified the peptides and their receptors from the 69 phylogenetic viewpoint using a newly established genetic database and bioinformatics. We 70 demonstrate, by phylogenetic and synteny analyses, that GRP is not a mammalian counterpart 71of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, 72the NMB/bombesin system has diversified in certain lineages, in particular in frogs. Gene 73 expression analyses combined with immunohistochemistry and Western blotting experiments 74indicate that GRP peptides and their receptors have evolved from ancestral (GRP) homologues 75to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems of 76 vertebrates, which is distinct from the frog bombesin lineage. 77 78( 165 words) 79 93 NMB-preferring receptor (NMBR or BB1 receptor) [10], and the potential orphan receptor, 94 bombesin receptor subtype-3 (BRS-3 or BB3 receptor) in mammals [11]. To date, in the 95 mammalian CNS, it has been reported that the GRP system might be integral, through 96 GRPR-mediated mechanisms [9], in a variety of autonomic-related functions, including food 97 intake [12-14], circadian rhythms [15-17], fear memory consolidation [18-20], male 98 reproductive function [21], control of sighing [22], and itch sensation [23, 24]. 99In birds, the expression of orthologous genes for GRP and GRPR has been reported 100 in the chicken CNS [25, 26]. To our knowledge, the only report of a central role of GRP in 101 avian behavior is decreased feeding after the intracerebroventricular administration of GRP in 102 chickens [27]. In amphibians, however, little information is currently available on the function 103 of the GRP/GRPR system in the CNS, because GRP has long been considered as the 104 mammalian equivalent of bombesin [5][6][7]. Thus, the central role of GRP in non-mammalian 105 species remains unclear. However, it has been reported that frogs synthesize both GRP and 106 bombesin, which are genetically distinct peptides, suggesting that GRP is not mammalian 107 bombesin. In addition, molecular cloning analyses revealed that 3 classes of receptor subtypes 108 were identified in the frog brain [28]. Based on amino acid sequence, two of the classes were 109 clearly the amphibian orthologous genes of the GRPR and NMBR, but not the BRS-3 [28].110 Moreover, a fourth class (BB4) of receptor from Bombina was identified in amphibians and, 111 interestingly, this had a higher binding affinity for bombesin than for either GRP or NMB 112 [28]. 113As described above, understanding...

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand its derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the ‘gut-brain peptide’ systems.

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