Functional discrimination between CRTH2 and DP. (A) DP but not CRTH2 mediates PGD₂-induced cAMP generation. Intracellular cAMP levels were determined by enzyme-linked immunoassay (mean, n = 2; Amersham Pharmacia Biotech). (B) CTX and PTX differentially affect PGD₂-induced (25 nM) Ca²+ mobilization in K562/CRTH2 and K562/DP. (C) Chemotactic responses to agonists (PGD₂, DK-PGD₂, and BW245C) are opposite in Jurkat/CRTH2 and Jurkat/DP (mean ± SD, n = 3). Numbers of migrated cells in controls were 727 ± 134 (Jurkat/CRTH2), 439 ± 50 (Jurkat/DP), and 661 ± 152 (Jurkat/neo).

A phylogenetic tree for human receptors to classical chemoattractants and major prostanoids. The tree was constructed by the N-J method using CLUSTAL X software. The sequence of the human α2B adrenergic receptor was used as an outer group to obtain a root. Chromosomal locations are shown in parentheses. Accession nos. for the receptors are (from top to bottom) GenBank/EMBL/DDBJ AF005900, D38081, L24470, L22647, L27490, L28175, D25418, U19487, Q13258 (GenPept accession no.), AF119711, AF254664, AB008193, AB008193, AB008535, M62505, L14061, L10820, and M84562. Cys LT, cysteinyl LT receptor; FPRL, formyl peptide receptor–like receptor.

Binding of PGD₂ to CRTH2. (A) Expression levels of CRTH2 as determined by flow cytometry. Cells were stained with biotinylated BM16 (solid line) or control IgG2a (dotted line) as described (reference 6). (B) CRTH2 mediates Ca²+ mobilization by PGD₂ and purified mast cell supernatants. Thrombin was used as an irrelevant stimulant. Arrows indicate the time of stimulant addition. (C) Scatchard plot analysis for ³H-PGD₂ binding to K562/CRTH2 or K562/DP. (D) CRTH2-mediated binding of ³H-PGD₂ is selectively inhibited by BM7. Cells pretreated with PBS, BM7 (600 μg/ml), or normal rat IgG (600 μg/ml) at room temperature for 20 min were subjected to ³H-PGD₂ binding analysis without washing (mean ± SD, n = 3). (E) DK-PGD₂ and BW245C serve as selective agonists for CRTH2 and DP, respectively, in Ca²+ mobilization assay.

Selective responses of Th2, basophils, and eosinophils to PGD₂. (A) CRTH2 expression in representative Th1 and Th2 lines. Levels of CRTH2 expression are presented as described in the legend to Fig. 1 A. (B) PGD₂ and DK-PGD₂ selectively induce Ca²+ mobilization in Th2 cells via CRTH2. Arrows indicate the time of addition of the test samples (black arrows) and 62.5 nM MCP-1 (white arrows). MCP-1 was used as an irrelevant stimulant. BM7 and rat IgG (each 600 μg/ml) were added 20 min before the stimulant addition. (C) Chemotaxis to PGD₂ and DK-PGD₂ is selectively induced in Th2 but not Th1 lines (mean ± SD, n = 3). The numbers of migrated cells in controls were 4,015 ± 627 (Th1) and 1,374 ± 225 (Th2). (D) Chemotaxis to PGD₂ and DK-PGD₂ of Th2 cells is inhibited by BM7 (mean ± SD, n = 3). Th2 cells were treated with PBS, BM7, or normal rat IgG as described in B before being subjected to chemotaxis assay. Numbers of migrated cells in controls were 4,146 ± 433 (PBS), 3,283 ± 349 (BM7), and 3,246 ± 65 (normal rat IgG). (E) mRNA levels for CRTH2, DP, and β-actin (internal control) in peripheral blood leukocytes and Th lines as determined by RT-PCR. (F) Migration of basophils and eosinophils is induced by PGD₂ (mean ± SD, n = 3). (G) Migration of eosinophils and basophils to PGD₂ and DK-PGD₂ is dose dependent (mean ± SD, n = 3). In this experiment, neutrophil (CD16⁺ cell)-depleted leukocyte samples were used. (H) PGD₂-induced migration of basophils and eosinophils is inhibited by BM7 (mean ± SD, n = 3). Neutrophil-depleted leukocytes were treated as described in D. Asterisks indicate statistical significance with a probability of <0.01 in Student's t test.