Lipid mediators are intercellular bioactive lipid molecules that induce various cellular responses mostly through G protein-coupled receptors (GPCRs). According to the IUPHAR database and recent publications, there are now approximately 50 lipid-recognizing GPCRs. Monitoring activation of GPCRs by lipid mediators or synthetic ligands is crucial for studying the actions of bioactive lipids as well as lipid GPCRs. Because each GPCR differentially couples with heterotrimeric G proteins (G s, G i/o, G q/11, G 12/13), in general it is necessary to prepare multiple GPCR assays to detect distinct G-protein signaling and individual optimization. In this protocol section, we describe a recently developed transforming growth factor-á (TGF-á) shedding assay, which is a simple and accurate method for measuring activation of lipid GPCRs. By utilizing coexpression of chimeric Gá subunits, the TGF-á shedding assay can detect activation of a wide range of GPCRs that are coupled with any of the four G proteins. Indeed, 39 of 45 (87 %) lipid GPCRs examined were detectable in the single format of the TGF-á shedding assay, demonstrating, to the best of our knowledge, the greatest coverage of a lipid GPCR assay. Thus, the TGF-á shedding assay provides a useful platform for analyzing lipid GPCRs.
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