CLONING AND EXPRESSION OF A PROSTAGLANDIN-E RECEPTOR EP(3) SUBTYPE FROM HUMAN ERYTHROLEUKEMIA-CELLS

Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP(3) subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP(3) receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the a subtype of the mouse EP(3) receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP(3) subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E(2) (PGE(2)) and binding PGE(2) with high specificity and a K-d of 3.2 nM. Radiolabelled PGE(2) could be displaced by prostaglandins in the order PGE(2) = PGE(1) > iloprost = PGD(2). Northern blot analysis revealed that the receptor is also present in human kidney.