Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.
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.
Zhuang
Hanyi
H
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA. hanyi.zhuang@duke.edu
Matsunami
Hiroaki
H
D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008156:Luciferases; D008967:Molecular Biology / Q000379:methods*; D018035:Receptors, Odorant / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D012903:Smell / Q000502:physiology*
D008024:Ligands; D018035:Receptors, Odorant; D008156:Luciferases
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