TGFα shedding assay: an accurate and versatile method for detecting GPCR activation

Nat Methods. 2012 Oct;9(10):1021-9. doi: 10.1038/nmeth.2172. Epub 2012 Sep 16.


A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) 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 Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) 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 Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • GTP-Binding Protein alpha Subunits, G12-G13 / physiology
  • GTP-Binding Protein alpha Subunits, Gq-G11 / physiology
  • HEK293 Cells
  • Humans
  • Lysophospholipids / metabolism
  • Receptors, G-Protein-Coupled / analysis*
  • Receptors, G-Protein-Coupled / physiology
  • Receptors, Purinergic P2 / metabolism
  • Signal Transduction
  • Transforming Growth Factor alpha / physiology*


  • Lysophospholipids
  • P2RY10 protein, human
  • Receptors, G-Protein-Coupled
  • Receptors, Purinergic P2
  • Transforming Growth Factor alpha
  • lysophosphatidylserine
  • GTP-Binding Protein alpha Subunits, G12-G13
  • GTP-Binding Protein alpha Subunits, Gq-G11