Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds

J Neurochem. 2006 Apr;97(2):537-44. doi: 10.1111/j.1471-4159.2006.03771.x. Epub 2006 Mar 15.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism
  • Cell Line
  • Cloning, Molecular / methods
  • Dose-Response Relationship, Drug
  • Flow Cytometry / methods
  • Gene Expression / drug effects
  • Gene Expression / physiology*
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Ligands
  • Models, Molecular
  • Odorants
  • Protein Structure, Secondary / physiology
  • Receptors, Odorant / chemistry*
  • Receptors, Odorant / metabolism*
  • Signal Transduction / drug effects
  • Stimulation, Chemical
  • Transfection / methods

Substances

  • Ligands
  • Receptors, Odorant
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Calcium