Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2
| Autor: | Terry P. Lybrand, Richard M. Breyer, Aaron N. Hata |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular Phenylalanine Indomethacin Receptors Prostaglandin Glutamic Acid Enzyme-Linked Immunosorbent Assay Ligands Binding Competitive Models Biological Biochemistry Cell Line Receptors G-Protein-Coupled Mice chemistry.chemical_compound Cell Movement Cyclic AMP Hypersensitivity Functional selectivity Enzyme-linked receptor Animals Humans Receptors Immunologic Receptor Molecular Biology Prostaglandin D2 receptor Inflammation Alanine Chemotactic Factors Dose-Response Relationship Drug Chemotaxis Ligand binding assay Anti-Inflammatory Agents Non-Steroidal Cell Biology Flow Cytometry Ligand (biochemistry) Receptor–ligand kinetics Protein Structure Tertiary Kinetics chemistry Mutation Mutagenesis Site-Directed Prostaglandins Eicosanoids Tyrosine lipids (amino acids peptides and proteins) Prostaglandin D2 Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 280:32442-32451 |
| ISSN: | 0021-9258 |
| Popis: | The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) 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 PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). 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 PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) 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 PGD(2) 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. |
| Databáze: | OpenAIRE |
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