IL-6

Authors

  • Franziska Noe Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Tim Frey Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Julia Fiedler Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Christiane Geithe Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Bettina Nowak Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut
  • Dietmar Krautwurst Deutsche Forschungsanstalt fuer Lebensmittelchemie - Leibniz Institut

DOI:

https://doi.org/10.14440/jbm.2017.206

Abstract

The assignment of cognate odorant/agonist pairs is a prerequisite for an understanding of odorant coding at the receptor level. However, the identification of new ligands for odorant receptors in cell-based assays has been challenging, due to their individual and rather sub-optimal plasma membrane expression, as compared with other G protein-coupled receptors. Accessory proteins, such as the chaperone RTP1S, or Ric8b, have improved the surface expression of at least a portion of odorant receptors. Typically, recombinant odorant receptors carry N-terminal tags, which proved helpful for their functional membrane expression. The most common tag is the ‘Rho-tag’, representing an N-terminal part of rhodopsin, but also ‘Lucy-‘ or ‘Flag-tag’ extensions have been described. Here, we used a bi-functional N-terminal tag, called ‘IL-6-HaloTag®’, with IL-6 facilitating functional cell surface expression of recombinant odorant receptors, and the HaloTag® protein, serving as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by live-cell flow cytometry. Our experiments revealed on average an about four-fold increased surface expression, a four-fold higher signaling amplitude, and a significant higher potency of odorant-induced cAMP signaling of six different human IL-6-HaloTag®-odorant receptors across five different receptor families in NxG 108CC15 cells, as compared to their Rho-tag–HaloTag® constructs. We observed similar results in HEK-293 cells. Moreover, screening an IL-6–HaloTag®-odorant receptor library with allyl phenyl acetate, revealed both known receptors as best responders for this compound. In summary, the IL-6–HaloTag® represents a promising tool for the de-orphaning of odorant receptors.

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Published

2017-11-03

How to Cite

1.
Noe F, Frey T, Fiedler J, Geithe C, Nowak B, Krautwurst D. IL-6. J Biol Methods [Internet]. 2017Nov.3 [cited 2021Dec.2];4(4):e81. Available from: https://jbmethods.org/jbm/article/view/206

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