Measuring Activation of Lipid G Protein- Coupled Receptors Using the TGF-á Shedding Assay

Asuka Inoue, Junken Aoki

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Lipid mediators are intercellular bioactive lipid molecules that induce various cellular responses mostly through G protein-coupled receptors (GPCRs). According to the IUPHAR database and recent publications, there are now approximately 50 lipid-recognizing GPCRs. Monitoring activation of GPCRs by lipid mediators or synthetic ligands is crucial for studying the actions of bioactive lipids as well as lipid GPCRs. Because each GPCR differentially couples with heterotrimeric G proteins (G s, G i/o, G q/11, G 12/13), in general it is necessary to prepare multiple GPCR assays to detect distinct G-protein signaling and individual optimization. In this protocol section, we describe a recently developed transforming growth factor-á (TGF-á) shedding assay, which is a simple and accurate method for measuring activation of lipid GPCRs. By utilizing coexpression of chimeric Gá subunits, the TGF-á shedding assay can detect activation of a wide range of GPCRs that are coupled with any of the four G proteins. Indeed, 39 of 45 (87 %) lipid GPCRs examined were detectable in the single format of the TGF-á shedding assay, demonstrating, to the best of our knowledge, the greatest coverage of a lipid GPCR assay. Thus, the TGF-á shedding assay provides a useful platform for analyzing lipid GPCRs.

Original languageEnglish
Title of host publicationBioactive Lipid Mediators
Subtitle of host publicationCurrent Reviews and Protocols
PublisherSpringer Japan
Pages393-408
Number of pages16
ISBN (Electronic)9784431556695
ISBN (Print)9784431556688
DOIs
Publication statusPublished - 2015 Jan 1

Keywords

  • Alkaline phosphatase (AP)
  • Bioactive lipid
  • Chimeric Gá subunit
  • HEK293 cells
  • Lipid mediator
  • P- Nitrophenylphosphate (p -NPP)
  • Protein-coupled receptor (GPCR)
  • TGF-á shedding assay
  • TNF-á-converting enzyme (TACE)

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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    Inoue, A., & Aoki, J. (2015). Measuring Activation of Lipid G Protein- Coupled Receptors Using the TGF-á Shedding Assay. In Bioactive Lipid Mediators: Current Reviews and Protocols (pp. 393-408). Springer Japan. https://doi.org/10.1007/978-4-431-55669-5_28