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 GRP 1-46, 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 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. 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. GRP 1-46 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. GRP 1-46 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, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.
Giraud
A S
AS
Murdoch Childrens Research Institute, Royal Childrens Hospital, Flemington Rd, Parkville, Victoria 3052, Australia. andrew.giraud@mcri.edu.au
Dumesny
C
C
Whitley
J C
JC
Parker
L M
LM
Jennings
I
I
Kemp
B
B
Moody
T W
TW
Sancho
V
V
Jensen
R T
RT
Shulkes
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004717:Endometrium / Q000737:chemistry*; D005260:Female; D019886:Gastrin-Releasing Peptide / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000235:genetics; D011247:Pregnancy; D011485:Protein Binding / Q000502:physiology; D011498:Protein Precursors / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D018004:Receptors, Bombesin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D007211:Indoles; D007295:Inositol Phosphates; C419604:PD 168368; D010455:Peptides; D011498:Protein Precursors; D011725:Pyridines; D018004:Receptors, Bombesin; C107120:bombesin receptor subtype 3; C047897:gastrin-releasing peptide precursor; D019886:Gastrin-Releasing Peptide; D010738:Type C Phospholipases