Abstract
A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.
Highlights
PGF receptortranfound in thecorpus luteum
TXA, PCR primers were designedbased on highly conservedcDNAsequences of the human and mouse receptors and mouse three PGE receptor subtypes corresponding to the putative second extracellular loop region and seventh transmembrane domain [14,15,16,17,18]
PGF, Binding and Inositol fiisphosphate (IPd FormationAssays in COS-I Cells-The cDNA insert of MC205 was subcloned into pcDNAI/ Amp (Invitrogen),and the resultantplasmid DNA was transfected into COS-1 cellsby the DEAE-dextran method [19].After culture for 72 h, the cells were harvested, and crude membranes were prepared as described [18]
Summary
(Received for publication, August 16, 1993, and in revised form, September 13, 1993). A functionalcDNAclone for mouse prostaglandin (PGbe)en carried out only poorly. For a clear understanding of the Freceptor was isolated froma mouse cDNA libraryus- phyiological rolesof PGF2a,we performed cDNA cloning of the ing polymerase chain reaction basoendthe sequence of PGF receptor. We report here the primary structure, ligand clonedprostanoidreceptors,andcross-hybridization binding and biochemical properties,and mRNA expression patscreening. ThemousePGFreceptor consists of 366 tern of the mouse PGF receptor.
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