Gastrin-releasing peptide (GRP) is not mammalian bombesin: Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.