Molecular cloning and characterization of the canine prostaglandin E receptor EP2 subtype.
Prostaglandin E2 (PGE2) binds to four G-protein coupled cell surface receptors (EP1-EP4) and has been implicated as a local mediator of bone anabolism via a cyclic AMP mediated pathway following activation of the EP2 and/or EP4 receptor subtype. A canine kidney cDNA library was screened using a human EP2 probe, and a clone with an open reading frame of 1083 bp, potentially encoding a protein of 361 amino acids, was characterized. This open reading frame has 89% identity to the human EP2 cDNA at the nucleotide level and 87% identity at the predicted protein level. Scatchard analysis of a CHO cell line stably transfected with canine EP2 yielded a dissociation constant of 22 nM for PGE2. Competition binding studies, using 3H-PGE2 as ligand, demonstrated specific displacement by PGE2, Prostaglandin E1, Prostaglandin A3, and butaprost (an EP2 selective ligand), but not by ligands with selectivity for the related DP, FP, IP, or TP receptors. Specific ligand binding also resulted in increased levels of cAMP in EP2 transfected cells with no evidence of short-term, ligand-induced desensitization. Northern blot analysis revealed two transcripts of 3300 and 2400 bp in canine lung, and reverse-transcription polymerase chain reaction showed expression in all tissues examined. Southern blot analysis suggests the presence of a single-copy gene for EP2 in the dog.
Department of Cardiovascular and Metabolic Diseases, Pfizer, Inc., Groton, Connecticut 06340, USA.
T A
TA
Hibbs
B
B
Lu
S L
SL
Smock
P
P
Vestergaard
L C
LC
Pan
T A
TA
Owen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015139:Blotting, Southern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004285:Dogs; D005786:Gene Expression Regulation; D008024:Ligands; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014162:Transfection
D008024:Ligands; C545644:PTGER2 protein, human; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D000242:Cyclic AMP; D015232:Dinoprostone