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


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2012-10

AUTHORS

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

ABSTRACT

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. More... »

PAGES

1021

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nmeth.2172

DOI

http://dx.doi.org/10.1038/nmeth.2172

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1001325351

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/22983457


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