Abstract
Prostaglandin D2 (PGD2), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD2 exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD2 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 [3H]PGD2, with a Kd 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-PGD2 (DK-PGD2) ≥ 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) ≥ PGD2 ≥ PGJ2. [3H]PGD2 binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a Ki value of 1.04 ± 0.13 μM. No [3H]PGD2 displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 μM. PGD2, DK-PGD2, 15d-PGJ2, 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 Gi heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD2, indomethacin, and 15d-PGJ2. mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on Gi and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.
Footnotes
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This work was supported by grants from the National Institutes of Health GM15431 (R.M.B.), DK46205 (R.M.B.), DK37097 (M.D.B.) as well as the Vanderbilt-Ingram Cancer Center. A.N.H. is supported by a PhRMA Foundation predoctoral fellowship. R.Z. is a clinician scientist of the National Kidney Foundation (NKF) and has an Advanced Career Development Award from the Veterans Administration.
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DOI: 10.1124/jpet.103.050955.
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ABBREVIATIONS: PGD2, prostaglandin D2; OVA, ovalbumin; GPCR, G-protein coupled receptor; [cAMP]i, intracellular cAMP; DK-PGD2, 13,14-dihydro-15-keto-PGD2; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; PPARγ, peroxisome proliferator-activated receptor-γ; IL, interleukin; NSAID, nonsteroidal anti-inflammatory drug; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; ponA, ponasterone A; PTX, pertussis toxin; PI 3-kinase, phosphatidylinositol 3-kinase; COX, cyclooxygenase; BW245C, (4S)-(3-[(R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptonoic acid; G418, geneticin.
- Received March 3, 2003.
- Accepted April 16, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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