HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 37, 32442-32451, September 16, 2005

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 280/37/32442    most recent M502563200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hata, A. N. Articles by Breyer, R. M. Search for Related Content PubMed PubMed Citation Articles by Hata, A. N. Articles by Breyer, R. M. Social Bookmarking                      What's this?

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2^*

From the 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

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.

Received for publication, March 8, 2005 , and in revised form, July 18, 2005.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by a Pharmaceutical and Research Manufacturers of America Foundation predoctoral fellowship.

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				I. M. Macias-Perez, R. Zent, M. Carmosino, M. D. Breyer, R. M. Breyer, and A. Pozzi Mouse EP3 {alpha}, {beta}, and {gamma} Receptor Variants Reduce Tumor Cell Proliferation and Tumorigenesis in Vivo J. Biol. Chem., May 2, 2008; 283(18): 12538 - 12545. [Abstract] [Full Text] [PDF]

				S. Basu, V. R. Jala, S. Mathis, S. T. Rajagopal, A. Del Prete, P. Maturu, J. O. Trent, and B. Haribabu Critical Role for Polar Residues in Coupling Leukotriene B4 Binding to Signal Transduction in BLT1 J. Biol. Chem., March 30, 2007; 282(13): 10005 - 10017. [Abstract] [Full Text] [PDF]