Expression of the Ghrelin Axis in the Mouse: An Exon 4-Deleted Mouse Proghrelin Variant Encodes a Novel C Terminal Peptide

Abstract

Ghrelin, an n-octanoylated 28-amino-acid peptide capable of inducing GH secretion and food intake in humans and rats, is the endogenous ligand for the GH secretagogue receptor (GHS-R). Here we describe the expression and tissue distribution of the ghrelin/GHS-R axis in the mouse. We also report for the first time the identification of a novel mouse ghrelin mRNA variant in which there is a complete deletion of exon 4. Translation of this variant mRNA yields a protein containing ghrelin and an alternative C-terminal domain with a unique C-terminal peptide sequence. RT-PCR with primers specific for mouse ghrelin was used to demonstrate the mRNA expression of the full preproghrelin transcript and the exon 4-deleted variant in multiple mouse tissues. Real-time PCR was also employed to quantitate mRNA expression of ghrelin, the novel isoform and a previously reported ghrelin gene variant, ghrelin gene-derived transcript. We also demonstrated the tissue expression of the functional GHS-R in the mouse. Immunohistochemistry, employing antibodies raised against the mature human n-octanoylated ghrelin peptide and the putative C-terminal peptide encoded by the exon 4-deleted proghrelin variant, was used to demonstrate protein expression of ghrelin and the variant in multiple mouse tissues including stomach, kidney, and reproductive tissues. The coexpression of ghrelin and its receptor in a wide range of murine tissues suggests varied autocrine/paracrine roles for these peptides. Exon 4-deleted proghrelin, a novel mouse proghrelin isoform with a unique C-terminal peptide sequence, is also widely expressed in the mouse and thus may possess biological activity in these tissues.

Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress

“…Consistent with the alternative usage of In2 observed in the current report, utilization of an alternative splice site in In2 has previously been reported to yield 'des-Gln 14 -ghrelin' in several species (Hosoda et al 2000b, Kawamura et al 2003Genbank #: AB089202). In addition, consistent with the current study, Jeffery et al (2005) recently reported the expression of a ghrelin variant that retains the coding sequence for mature ghrelin but has a novel C-terminal tail due to the exclusion of Ex4 resulting in a cDNA frameshift that generates a premature stop codon. The same group previously identified a human homolog of this isoform that is expressed in a wide range of human tissues (Jeffery et al 2002).…”

Ghrelin, des-acyl ghrelin and obestatin: Three pieces of the same puzzle

“…A murine homologue (exon 4-deleted mRNA transcript) has also been identified in a widely range of murine tissues [75]. It is probable that theses variants, like the pre-prodes-Gln14-ghrelin mRNA, are products of the ghrelin gene produced by alternative splicing mechanisms.…”

“…Preproghrelin contains a 23 amino-acid signal peptide and a 94 amino-acid called pro-ghrelin . This includes the 28 amino acid mature ghrelin (1-28) and a 66 amino acid tail [83,75]. The enzymes responsible for proteolytic cleavage of preproghrelin to the final products remain largely unknown.…”

“…In fact, numerous studies have shown that ghrelin also affects energy and glucose homeostasis, gastrointestinal, cardiovascular, pulmonary and immune function, cell proliferation and differentiation and bone physiology [84,91,150]. Although the major active product of ghrelin gene is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group (C8:0), called simply ghrelin, recent developments have shown that ghrelin gene can generate various bioactive molecules besides ghrelin, mainly des-acyl ghrelin and obestatin, obtained from alternative splicing or from extensive post-translational modifications [69,70,75,160,162]. Although their receptors have not yet been identified, they have already proven to be active, having intriguingly subtle but opposite physiological actions to ghrelin [84,91,150,162].…”

“…It comprises five exons and three introns, being the short first exon only 20 bp long, which encode part of the 5 0 untranslated region [77,81]. Mature ghrelin (i.e., the 28-amino acid GH releasing peptide) is encoded by exons 2 and 3 with the remaining proghrelin peptide sequence contained in exons 4 and 5 [75]. The putative initiation codon ATG is located at nucleotides 34-36, preceded by the consensus initiation sequence, whereas a terminal codon TAG is found 117 codons downstream at position 385-387 [75].…”