A bi-functional IL-6-HaloTag® as a tool to measure the cell-surface expression of recombinant odorant receptors and to facilitate their activity quantification

Main Article Content

Franziska Noe
Christiane Geithe
Julia Fiedler
Dietmar Krautwurst

Keywords

De-orphaning, G protein-coupled receptor, GPCR, HEK-293, NxG 108CC15

Abstract

The functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, “tagged” receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal ‘IL-6-HaloTag®’. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag®-odorant receptors, and the HaloTag® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag®-odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor® cAMP assay.

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References

1. Buck L, Axel R (1991) A novel multigene family may encode odorant receptors - a molecular-basis for odor recognition. Cell 65: 175-187. doi: 10.1016/0092-8674(91)90418-X. PMID: 1840504
2. Krautwurst D, Yau KW, Reed RR (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95: 917-926. doi: 10.1016/S0092-8674(00)81716-X. PMID: 9875846
3. Zhao H, Ivic L, Otaki JM, Hashimoto M, Mikoshiba K, et al. (1998) Functional expression of a mammalian odorant receptor. Science 279: 237-242. doi: 10.1126/science.279.5348.237. PMID: 9422698
4. Reed RR (1992) Signaling pathways in odorant detection. Neuron 8: 205-209. doi: 10.1016/0896-6273(92)90287-N. PMID: 1739458
5. Touhara K, Vosshall LB (2009) Sensing odorants and pheromones with chemosensory receptors. Annu Rev Physiol 71: 307-332. doi: 10.1146/annurev. physiol.010908.163209. PMID: 19575682
6. Bushdid C, Magnasco MO, Vosshall LB, Keller A (2014) Humans can discriminate more than 1 trillion olfactory stimuli. Science 343: 1370-1372. doi: 10.1126/science.1249168. PMID: 24653035
7. Dunkel A, Steinhaus M, Kotthoff M, Nowak B, Krautwurst D, et al. (2014) Nature's chemical signatures in human olfaction: a foodborne perspective for future biotechnology. Angew Chem Int Ed Engl 53: 7124-7143. doi: 10.1002/anie.201309508. PMID: 24939725
8. Malnic B, Hirono J, Sato T, Buck LB (1999) Combinatorial receptor codes for odors. Cell 96: 713-723. PMID: 10089886
9. Ghiringhelli C, Ghiringhelli L, Redaelli G, Crespi B (1992) [Skin versus mesh in the treatment of laparoceles]. Minerva Chir 47: 419-422. doi: 10.1111/j.1471-4159.2004.02619.x. PMID: 1534152
10. McClintock TS, Sammeta N (2003) Trafficking prerogatives of olfactory receptors. Neuroreport 14: 1547-1552. doi: 10.1097/01.wnr.0000085904.20980.e1. PMID: 14502073
11. Zhuang H, Matsunami H (2007) Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem 282: 15284-15293. doi: 10.1074/jbc.M700386200. PMID: 17387175
12. Geithe C, Andersen G, Malki A, Krautwurst D (2015) A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors. J Agric Food Chem 63: 9410-9420. doi: 10.1021/acs.jafc.5b01884. PMID: 26451762
13. Shirokova E, Schmiedeberg K, Bedner P, Niessen H, Willecke K, et al. (2004) Identification of specific ligands for orphan olfactory receptors. G proteindependent agonism and antagonism of odorants. J Biol Chem 280: 11807-11815. doi: 10.1074/jbc.M411508200. PMID: 15598656
14. Touhara K (2007) Deorphanizing vertebrate olfactory receptors: recent advances in odorant-response assays. Neurochem Int 51: 132-139. doi: 10.1016/j.neuint.2007.05.020. PMID: 17640771
15. Zhuang H, Matsunami H (2008) Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc 3: 1402-1413. doi: 10.1038/nprot.2008.120. PMID: 18772867
16. Binkowski B, Fan F, Wood K (2009) Engineered luciferases for molecular sensing in living cells. Curr Opin Biotechnol 20: 14-18. doi: 10.1016/j.copbio.2009.02.013. PMID: 19299118
17. Urh M, Rosenberg M (2012) HaloTag, a Platform Technology for Protein Analysis. Curr Chem Genomics 6: 72-78. doi: 10.2174/1875397301206010072. PMID: 23213345
18. Assier E, Boissier M, Dayer J (2010) Interleukin-6: from identification of the cytokine to development of targeted treatments. Joint Bone Spine 77: 532-536. doi: 10.1016/j.jbspin.2010.07.007. PMID: 20869898
19. Noe F, Frey T, Fiedler J, Geithe C, Nowak B, et al. (2017). IL-6–HaloTag® enables live-cell plasma membrane staining, flow cytometry, functional expression, and de-orphaning of recombinant odorant receptors. J Biol Methods 4: e81. doi: 10.14440/jbm.2017.206.
20. Hamprecht B, Glaser T, Reiser G, Bayer E, Propst F (1985) Culture and characteristics of hormone-responsive neuroblastoma X glioma hybrid cells. Methods Enzymol 109: 316-341. PMID: 2985920
21. Reisert J (2010) Origin of basal activity in mammalian olfactory receptor neurons. J Gen Physiol 136: 529-540. doi: 10.1085/jgp.201010528. PMID: 20974772

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