Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

Abstract

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant–OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.

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Figure 1: Overview of the functional activation of ORs in a heterologous expression system.
Figure 2: The endogenous and heterologous OR signal transduction pathways.
Figure 3
Figure 4: An example of cell-surface OR expression.
Figure 5: An example of cell-surface flow cytometry.
Figure 6: A hypothetical case of luciferase assay data interpretation.

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Acknowledgements

We thank A. Toyama for FACS data. This research was supported by grants to H.M. from the NIH and Human Frontier Science Program and to H.Z. from an NIH NRSA Predoctoral fellowship.

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Correspondence to Hanyi Zhuang or Hiroaki Matsunami.

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Zhuang, H., Matsunami, H. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc 3, 1402–1413 (2008). https://doi.org/10.1038/nprot.2008.120

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