Isolation, identification and biological activity of gastrin-releasing peptide 1-46 (oGRP1-46), the primary GRP gene-derived peptide product of the pregnant ovine endometrium

Andrew Giraud, C Dumesny, Jane Whitley, L Parker, Ian Jennings, Bruce Kemp, Terry Moody, Veronica Sancho, RT Jensen, Arthur Shulkes

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Abstract

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125a??140 day pregnant sheep uterus (term is 145 day) was homogenized in 80 acetonitrile/2 trifluoroacetic acid (1:7 ACN/TFA), concentrated on reversephase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 I?Bondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP1-46 would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP18-27 and GRP 1-27 in other tissues. GRP1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.
Original languageEnglish
Pages (from-to)284 - 290
Number of pages6
JournalPeptides
Volume31
Issue number2
DOIs
Publication statusPublished - 2010

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