Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds.
Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17-40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca(2+) ion flux allowed dose-dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17-40-specific antagonists. The C-terminal fusion of enhanced green fluorescent protein to the hOR17-40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17-40 cell surface targeting and odorant-evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence-activated cell sorting was used to separate hOR17-40-expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant-responsive cells to up to 80% of the total cell number.
Jacquier
Valérie
V
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland.
Pick
Horst
H
Vogel
Horst
H
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009812:Odorants; D017433:Protein Structure, Secondary / Q000502:physiology; D018035:Receptors, Odorant / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013268:Stimulation, Chemical; D014162:Transfection / Q000379:methods
D008024:Ligands; D018035:Receptors, Odorant; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium