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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References
Vassilatis, D.K. et al. The G protein-coupled receptor repertoires of human and mouse. Proc. Natl. Acad. Sci. USA 100, 4903–4908 (2003).
Downes, G.B. & Gautam, N. The G protein subunit gene families. Genomics 62, 544–552 (1999).
Inoue, A. et al. LPA-producing enzyme PA-PLA(1)alpha regulates hair follicle development by modulating EGFR signalling. EMBO J. 30, 4248–4260 (2011).
Edwards, D.R., Handsley, M.M. & Pennington, C.J. The ADAM metalloproteinases. Mol. Aspects Med. 29, 258–289 (2008).
Rasmussen, S.G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D. & Bourne, H.R. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363, 274–276 (1993).
Conklin, B.R. et al. Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. Mol. Pharmacol. 50, 885–890 (1996).
Liu, J., Conklin, B.R., Blin, N., Yun, J. & Wess, J. Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation. Proc. Natl. Acad. Sci. USA 92, 11642–11646 (1995).
Offermanns, S. & Simon, M.I. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175–15180 (1995).
Ehlert, F.J. Analysis of allosterism in functional assays. J. Pharmacol. Exp. Ther. 315, 740–754 (2005).
Kobilka, B.K. & Deupi, X. Conformational complexity of G-protein-coupled receptors. Trends Pharmacol. Sci. 28, 397–406 (2007).
Bond, R.A. & Ijzerman, A.P. Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol. Sci. 27, 92–96 (2006).
Sugo, T. et al. Identification of a lysophosphatidylserine receptor on mast cells. Biochem. Biophys. Res. Commun. 341, 1078–1087 (2006).
Murakami, M., Shiraishi, A., Tabata, K. & Fujita, N. Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor. Biochem. Biophys. Res. Commun. 371, 707–712 (2008).
Wong, S.K. G protein selectivity is regulated by multiple intracellular regions of GPCRs. Neurosignals 12, 1–12 (2003).
Alexander, S.P., Mathie, A. & Peters, J.A. Guide to receptors and channels (GRAC), 3rd edn. British J. Pharmacol. 153 (suppl. 2), S1–S209 (2008).
Noguchi, K., Ishii, S. & Shimizu, T. Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J. Biol. Chem. 278, 25600–25606 (2003).
Chun, J., Hla, T., Lynch, K.R., Spiegel, S. & Moolenaar, W.H. International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol. Rev. 62, 579–587 (2010).
Makide, K., Kitamura, H., Sato, Y., Okutani, M. & Aoki, J. Emerging lysophospholipid mediators, lysophosphatidylserine, lysophosphatidylthreonine, lysophosphatidylethanolamine and lysophosphatidylglycerol. Prostaglandins Other Lipid Mediat. 89, 135–139 (2009).
Iwashita, M. et al. Synthesis and evaluation of lysophosphatidylserine analogues as inducers of mast cell degranulation. Potent activities of lysophosphatidylthreonine and its 2-deoxy derivative. J. Med. Chem. 52, 5837–5863 (2009).
Zhao, Z. & Xu, Y. Measurement of endogenous lysophosphatidic acid by ESI-MS/MS in plasma samples requires pre-separation of lysophosphatidylcholine. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 3739–3742 (2009).
Sato, T. et al. Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family. J. Biol. Chem. 272, 2192–2198 (1997).
Nakamura, K. et al. A novel enzyme immunoassay for the determination of phosphatidylserine-specific phospholipase A(1) in human serum samples. Clinica Chimica Acta 411, 1090–1094 (2010).
Liebscher, I. et al. Altered immune response in mice deficient for the G protein-coupled receptor GPR34. J. Biol. Chem. 286, 2101–2110 (2011).
Peschon, J.J. et al. An essential role for ectodomain shedding in mammalian development. Science 282, 1281–1284 (1998).
Shimizu, Y. et al. ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J. Cell Biol. 168, 941–953 (2005).
Thumkeo, D., Shimizu, Y., Sakamoto, S., Yamada, S. & Narumiya, S. ROCK-I and ROCK-II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo. Genes Cells 10, 825–834 (2005).
Canault, M., Certel, K., Schatzberg, D., Wagner, D.D. & Hynes, R.O. The lack of ADAM17 activity during embryonic development causes hemorrhage and impairs vessel formation. PLoS ONE 5, e13433 (2010).
Offermanns, S., Mancino, V., Revel, J.P. & Simon, M.I. Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency. Science 275, 533–536 (1997).
Blaydon, D.C. et al. Inflammatory skin and bowel disease linked to ADAM17 deletion. N. Engl. J. Med. 365, 1502–1508 (2011).
Gelling, R.W. et al. Deficiency of TNFalpha converting enzyme (TACE/ADAM17) causes a lean, hypermetabolic phenotype in mice. Endocrinology 149, 6053–6064 (2008).
Tokumaru, S. et al. Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing. J. Cell Biol. 151, 209–220 (2000).
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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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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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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DOI: https://doi.org/10.1038/nmeth.2172
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