TGF[alpha] shedding assay: an accurate and versatile method for detecting GPCR activation

A single-format method to detect multiple G protein–coupled receptor (GPCR) signaling, especially Gα12/13 signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase–tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα12/13 and Gαq signaling. Relying on chimeric Gα proteins and promiscuous Gα16 protein, which can couple with Gαs- and Gαi-coupled GPCRs and induce Gαq signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα12/13-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα12/13-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs. Download references