Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2*
- Departments of Pharmacology, Medicine, Chemistry, the Vanderbilt Ingram Cancer Center, and the Vanderbilt Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
- 2 To whom correspondence should be addressed: Division of Nephrology, Vanderbilt University School of Medicine, S3223 MCN, 1161 21st Ave., Nashville, TN 37232-2372. Tel.: 615-343-0257; Fax: 615-343-4704; E-mail: rich.breyer{at}vanderbilt.edu.
Abstract
The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D2 (PGD2) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD2 binding affinity, suggesting that these residues play a role in binding PGD2. In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD2, whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE2 by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD2 binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.
Footnotes
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↵3 The abbreviations used are: PGD2, prostaglandin D2; C5aR, C5a anaphylatoxin receptor; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; DK-PGD2, 13,14-dihydro-15-keto-PGD2; DP, D prostanoid; EC, extracellular loop domain; FPR, formyl peptide receptor; GPCR, G protein-coupled receptor; NSAID, nonsteroidal anti-inflammatory drug; TM, transmembrane domain; IP, prostacyclin receptor; HA, hemagglutinin; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FBS, fetal bovine serum.
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↵* This work was supported in part by National Institutes of Health Grants AI59108 (to R. M. B.), GM15431 (to R. M. B.), DK46205 (to R. M. B.), and NS33290 (to T. P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
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↵1 Supported by a Pharmaceutical and Research Manufacturers of America Foundation predoctoral fellowship.
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- Received March 8, 2005.
- Revision received July 18, 2005.
- The American Society for Biochemistry and Molecular Biology, Inc.
