Expression and molecular pharmacology of the mouse CRTH2 receptor

Prostaglandin D-2 (PGD(2)), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD(2) exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD(2) mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [H-3]PGD(2), with a K-d of 8.8 +/- 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD(2) (DK-PGD(2)) greater than or equal to 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) greater than or equal to PGD(2) greater than or equal to PGJ(2). [H-3]PGD(2) binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a K-i value of 1.04 +/- 0.13 muM. No [H-3]PGD(2) displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 muM. PGD(2), DK-PGD(2), 15d-PGJ(2), and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a G(i) heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD(2), indomethacin, and 15d-PGJ(2). mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on G(i) and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.