Expression and Characterization of a 5-oxo-6E,8Z,11Z,14Z-Eicosatetraenoic Acid Receptor Highly Expressed on Human Eosinophils and Neutrophils

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Abstract
Using a bioinformatics approach, we have isolated a novel G-protein-coupled receptor (GPCR), R527, and have demonstrated that this receptor shows no significant homology to previously deorphanized GPCRs. Quantitative reverse transcription-polymerase chain reaction analysis of the expression of GPCR R527 indicated a very high level of mRNA expression in eosinophils, with high expression also detected in neutrophils and lung macrophages. Stable cell lines were generated expressing this receptor together with the G-protein α-subunit Gα16. These cells were used to screen an agonist collection in a calcium mobilization assay and 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) was identified as a putative ligand. 5(S)-Hydroxyperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid was also shown to activate the receptor, whereas the leukotrienes LTB4, LTC4, LTD4, and LTE4 failed to elicit a response. In cAMP assays, pertussis toxin reversed the inhibitory effects of 5-oxo-ETE on cAMP production, indicating that the receptor is Gαi-coupled. The GPCR R527 shows pharmacological properties similar to those of the previously described 5-oxo-ETE receptor expressed on eosinophils, neutrophils, and monocytes. These cell types show chemotactic responses to 5-oxo-ETE, and this eicosanoid has been proposed to play a key role in the inflammatory response. The molecular identification of a receptor binding 5-oxo-ETE will expand our understanding of the physiological role of this mediator and may provide new therapeutic opportunities.