TGFα 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 G alpha(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-alpha (TGF alpha) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGF alpha (AP-TGF alpha) and its release into conditioned medium. AP-TGF alpha shedding response occurred almost exclusively downstream of G alpha(12/13) and G alpha(q) signaling. Relying on chimeric G alpha proteins and promiscuous G alpha(16) protein, which can couple with G alpha(s)-and G alpha(i)-coupled GPCRs and induce G alpha(q) signaling, we used the TGF alpha shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as G alpha(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGF alpha shedding assay is useful for studies of poorly characterized G alpha(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.