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

Asuka Inoue, Jun Ishiguro, Hajime Kitamura, Naoaki Arima, Michiyo Okutani, Akira Shuto, Shigeki Higashiyama, Tomohiko Ohwada, Hiroyuki Arai, Kumiko Makide, Junken Aoki

Research output: Contribution to journalArticlepeer-review

244 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)1021-1029
Number of pages9
JournalNature Methods
Issue number10
Publication statusPublished - 2012 Oct

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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