Molecular cloning and functional characterization of the canine prostaglandin E2 receptor EP4 subtype.

Prostaglandin E2 (PGE2) is an important mediator of diverse biologic functions in many tissues and binds with high affinity to four cell surface, seven-transmembrane domain, G protein-coupled receptors (EP1-EP4). The EP4 receptor subtype has a long intracellular carboxy-terminal region and is functionally coupled to adenylate cyclase, resulting in elevated intracellular cyclic adenosine 5' monophosphate (cAMP) levels upon activation. To further study EP4 receptor subtype function, a canine kidney cDNA library was screened and three clones were isolated and sequenced. The longest clone was 3,103 bp and contained a single open reading frame of 1,476 bp, potentially encoding a protein of 492 amino acids with a predicted molecular weight of 53.4 kDa. Sequence analysis of this open reading frame reveals 89% identity to the human EP4 protein coding region at the nucleotide level and 90% identity when the putative canine and human protein sequences are compared. Northern blot analysis showed relatively high levels of canine EP4 expression in heart, lung and kidney, while Southern blot analysis of canine genomic DNA suggests the presence of a single copy gene. Following transfection of canine EP4 into CHO-KI cells, Scatchard analysis revealed a dissociation constant of 24 nM for PGE, while competition binding studies using 3H-PGE2 as ligand demonstrated specific displacement by PGE2 prostaglandin E, (PGE1), and prostaglandin A3 (PGA3). Treatment with PGE2 also resulted in increased levels of cAMP in transfected, but not in parental, CHO-KI cells. In contrast, butaprost, an EP2 selective ligand, and sulprostone, an EP1/EP3 selective ligand, did not bind to this receptor at the maximal concentration used (320 nM). To further investigate secondary signaling, the canine EP4 cDNA was truncated to produce an 1,117 bp fragment encoding a 356 amino acid protein lacking the intracellular carboxy-terminus. When transfected, this truncated cDNA produced a protein with a dissociation constant of 11 nM for PGE2 and a binding and cAMP accumulation profile similar to that of the full-length protein. Both full-length and truncated canine EP4 underwent short term PGE2-induced desensitization as shown by a lack of continuing cAMP accumulation after the initial PGE2 stimulation, suggesting no involvement of the C-terminal intracellular tail. This result is in contrast to that reported for the human EP4 receptor, where residues within the C-terminal intracellular tail were shown to mediate short term, ligand induced desensitization.