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

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 125–140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 μBondapak). 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 GRP 1–46 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 GRP 18–27 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.