SPLICE VARIANTS OF THE HUMAN EP(3) RECEPTOR FOR PROSTAGLANDIN E(2)

The EP(3) receptor for prostaglandin E(2) (PGE(2)) mediates various bi ological activities such as uterine contraction, inhibition of gastric acid secretion, presynaptic inhibition of neurotransmitter release an d potentiation of platelet aggregation. In an attempt to understand th e molecular basis of this diversity of biological function, we cloned full-length cDNAs encoding EP(3) receptors for PGE(2) from human uteru s cDNA libraries. Seven cDNA variants were identified which code for s ix distinct EP(3)-receptor isoforms. Sequencing revealed that the rece ptor isoforms differ in their intracellular C-terminal domains. Southe rn blot experiments indicate that the isoforms are generated by altern ative splicing. The EP(3)-receptor gene is expressed in various tissue s with high expression in kidney and pancreas, as demonstrated by Nort hern blot analysis. All receptors, stably expressed in baby hamster ki dney (BHK) cells, bind PGE(2) specifically with similar K-d of 2.2-5.8 nM. The binding of [H-3]PGE(2) is competed with by unlabelled prostag landins in the order sulprostone (a PGE(2)-like agonist) approximate t o PGE(2) much greater than PGF(2 alpha) > Iloprost (a prostacyclin ana logue) > PGD(2), which is specific for EP(3) receptors. Analysis of th e signal-transduction pathways demonstrated that all receptors respond with inhibition of forskolin-induced cAMP accumulation with an IC50 o f 0.13 nM PGE(2). In addition, some isoforms induce an increase in int racellular free calcium ([Ca2+](i)) at PGE(2) concentrations greater t han or equal to 10 nM. These results may offer an explanation for the different physiological responses observed in various tissues followin g activation of E(3) receptors.