Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia 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 EP3 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 EP3 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 alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.
Kunapuli
S P
SP
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Fen Mao
G
G
Bastepe
M
M
Liu-Chen
L Y
LY
Li
S
S
Cheung
P P
PP
DeRiel
J K
JK
Ashby
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D011453:Prostaglandins / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011453:Prostaglandins; D018078:Receptors, Prostaglandin E; D000242:Cyclic AMP