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

Nature Methods volume 9, pages 1021–1029 (2012)Cite this article

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Abstract

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.

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Figure 1: The principle and data processing of the TGFα shedding assay.
Figure 2: Gαq-coupled and Gα12/13-coupled GPCRs efficiently induce AP-TGFα release.
Figure 3: GPCR-induced AP-TGFα release is mainly dependent on Gαq and Gα12/13 signaling.
Figure 4: Extended TGFα shedding assay using chimeric Gα proteins and promiscuous Gα16 protein.
Figure 5: Pharmacological evaluation of GPCR ligands in the TGFα shedding assay.
Figure 6: P2Y10, A630033H20 and GPR174 identified as Gα12/13-coupled LysoPS-specific receptors.

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Acknowledgements

We thank N. Nakahata and M. Saito (Graduate School of Pharmaceutical Sciences, Tohoku University, Japan) for technical advice for developing the TGFα shedding assay and M. Arita (Graduate School of Pharmaceutical Sciences, the University of Tokyo) for providing plasmids encoding GPCRs. J.A. was supported by grants from the National Institute of Biomedical Innovation of Japan, the National Project on Protein Structural and Functional Analyses Grant-in-Aid for Scientific Research on Innovative Areas (KAKENHI 22116004) from the Ministry of Education, Science, Sports and Culture of Japan (MEXT) and (Precursory Research for Embryonic Science and Technology PRESTO) from Japan Science and Technology Agency. I.A. was funded by a Grant-in-Aid for Young Scientists (B) (KAKENHI 21790058).

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Authors and Affiliations

  1. Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

    Asuka Inoue, Jun Ishiguro, Hajime Kitamura, Naoaki Arima, Michiyo Okutani, Akira Shuto, Kumiko Makide & Junken Aoki

  2. Proteo-Medicine Research Center, Ehime University, Ehime, Japan

    Shigeki Higashiyama

  3. Department of Biochemistry and Molecular Genetics, Graduate School of Medicine, Ehime University, Ehime, Japan

    Shigeki Higashiyama

  4. Laboratory of Organic and Medicinal Chemistry, The University of Tokyo, Tokyo, Japan

    Tomohiko Ohwada

  5. Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Hiroyuki Arai

  6. Japan Science and Technology Agency, Core Research for Evolutional Sciences and Technology (CREST), Saitama, Japan

    Hiroyuki Arai & Junken Aoki

  7. Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), Saitama, Japan

    Kumiko Makide

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  1. Asuka Inoue
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Contributions

A.I. designed studies. A.I. and J.I. performed most of the experiments and analyzed data. N.A. and S.H. contributed to the development of the assay. H.K., N.A., M.O., A.S. and K.M. performed screening of orphan GPCRs. T.O. and H.A. synthesized LysoPS analogs. A.I. and J.A. prepared the paper.

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Correspondence to Asuka Inoue or Junken Aoki.

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The authors declare no competing financial interests.

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Supplementary Figures 1–20, Supplementary Tables 1–9, Supplementary Protocol (PDF 26336 kb)

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Inoue, A., Ishiguro, J., Kitamura, H. et al. TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. Nat Methods 9, 1021–1029 (2012). https://doi.org/10.1038/nmeth.2172

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