Molecular cloning and expression of a cDNA of the bovine prostaglandin F2 alpha receptor.
Capitalizing on the significant sequence homology comprising the transmembrane motif regions of known prostanoid receptor family, we targeted the cloning of a cDNA clone for prostaglandin (PG) F2 alpha receptor from a bovine corpus luteum cDNA library. By using several pairs of degenerated primers created from a common motif of transmembrane domains, polymerase chain reaction gave a clone SN463 carrying the homologous sequence, which covered transmembrane motif IV-VI of the thromboxane (TX) A2 receptor. This polymerase chain reaction product was used as a DNA probe for the following cross-hybridization, and a clone BC2211 carrying a 2.2-kilobase pair DNA insert was isolated. This clone encodes a protein of 362 amino acid residues (M(r) = 40,983) with seven potential transmembrane domains and represented significant overall sequence homology to human TXA2 receptor protein (34% in amino acid). Injection of the mRNA synthesized in vitro from the cloned cDNA into a Xenopus oocyte elicited electrophysiological response to PGF2 alpha. Ligand binding displacement in membranes of mammalian COS-7 cells transfected with the cDNA indicated the rank order of affinity of the receptor to PGs: PGF2 alpha > PGD2 > PGE2 > STA2, a TXA2 agonist. PGF2 alpha activated inositol phosphate formation in COS-7 cells transfected with receptor cDNA. Northern blot analysis and in situ hybridization indicated that the PGF2 alpha receptor mRNA is highly expressed and accumulated in corpus luteum. This is the first report on a successful cloning of functional receptor cDNA for PGF2 alpha.
Sakamoto
K
K
Department of Cell Biology, Osaka Bioscience Institute, Suita, Japan.
Ezashi
T
T
Miwa
K
K
Okuda-Ashitaka
E
E
Houtani
T
T
Sugimoto
T
T
Ito
S
S
Hayaishi
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003338:Corpus Luteum / Q000378:metabolism; D017931:DNA Primers; D015237:Dinoprost / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014981:Xenopus
D017931:DNA Primers; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D011061:Poly A; D012313:RNA; D015237:Dinoprost