Probing Odorant-Mediated Receptor Activation

Molecular interactions of odorants with their olfactory receptors (ORs) are of central importance for the mammalian olfactory system to detect and discriminate a large variety of odors with a limited set of receptors. How a particular OR binds and distinguishes different odorant molecules remains unknown on a structural basis as there is no three- dimensional OR structure available. Progress in the area of ligand assignment to specific receptors with the development of functional assays allowing efficient screening of numerous odorant compound libraries is essential for the understanding of the selectivity and sensitivity of odor detection. To improve our knowledge of an OR binding site environment, the first part of this thesis focused on the characterization of the molecular interactions of the mouse eugenol receptor (mOR-EG) which has been shown to be activated by a wide variety of odorants. Computer modeling based on the resolved 3D structure of the β2-adrenergic receptor and mutational studies on mOR-EG were used to determine the key amino acid residues responsible for specific OR-ligand recognition and to determine the localization and chemical properties of the putative ligand binding site. Activation of mOR-EG point mutants was monitored using a cAMP-induced reporter gene assay in Hana3A cells. These investigations were used to improve ligand docking in the binding site and further define a more precise model of the mOR-EG ligand-binding cavity. With the development of genome sequencing, the comparison of ortholog/paralog pairs is becoming increasingly important to study the molecular basis of OR specificity. Emphasis has thus been placed on the characterization of the mOR-EG’s mouse paralog mOR-EV and on its putative human ortholog hOR5D18. These two ORs have been functionally expressed in Hana3A cells and the visualization of both their overall- and cell-surface expression have been performed using different OR fusion proteins. New specific agonists for the mOR-EV have been identified (jasmone, rose oxyde and nootkatone) and first attempts to de-orphanize hOR5D18 have been carried out, by random screening a large panel of more than 250 odorant compounds coupling the OR activation to cAMP-induced reporter gene expression. Based on our knowledge of the mOR-EG ligand binding site, three important positions for ligand recognition that diverge between mOR-EG and mOR-EV were mutated in both receptors to increase their sequence identity. Comparison of their respective ligand repertoires has provided novel insight into the molecular determinants for odor detection and discrimination. Given the importance to assign ligands to their cognate receptors, we established a novel functional assay based on electrical impedance measurements. The detection of ligand-induced cell morphological changes during and after receptor stimulation has been used to quantify the activation of different GPCRs including mOR-EG with their respective ligands. This label-