The Mouse Eugenol Odorant Receptor: Structural and Functional Plasticity of a Broadly Tuned Odorant Binding Pocket

Molecular interactions of odorants with their olfactory receptors (ORs) are of central importance for the ability of the mammalian olfactory system to detect and discriminate a vast variety of odors with a limited set of receptors. How a particular OR binds and distinguishes different odorant molecules remains largely unknown on a structural basis. Here we investigated this question for the mouse eugenol receptor (mOR-EG). By screening a large odorant library, we discovered a wide range of chemical structures activating the receptor in heterologous mammalian cells. Potent agonists comprise (i) benzene, (ii) cyclohexane, or (iii) polycyclic structures substituted with alcohol, aldehyde, keto, ether, or esterified carboxylic groups. To detect those amino acids within the receptor that are in contact with a particular bound odorant molecule, we investigated how distinct mOR-EG point mutants were activated by the different odorant agonists found for the wild-type receptor. We identified 11 amino acids as a part of the receptor's ligand binding pocket. Molecular modeling predicted 10 of these residues in transmembrane helices TM3-TM6 and one in the extracellular loop between TM2 and TM3. These amino acids participate in odorant binding with variable importance depending on the type of odorant, revealing functional "fingerprints" of ligand-receptor interactions.

Probing Odorant-Mediated Receptor Activation

Olivia Baud

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-free technique is applicable for many different cell types as well as for endogenously expressed receptors and is suitable for screening large libraries of ORs and compounds. Odorants are generally small hydrophobic cell-membrane-permeable organic molecules with highly variable chemical structures and properties. Due to their large chemical diversity, it is likely that some of them might trigger other cellular processes apart from their "conventional" role in olfaction. The dual role of some odorants acting on the mOR-EG and/or the nuclear estrogen receptor α (ERα) has therefore been explored. Estrogenic effect of these odorants has been measured on ER endogenously expressing MCF7 cells, which represent a sensitive and well-established system for the detection of estrogenic activities, using a Luciferase reporter gene assay and different cell proliferation measuring methods. Finally, computational modeling and ligand docking have provided informations on the molecular recognition principles of odorants on two functionally non-related receptors and will therefore help for the evaluation of the potential risk of chemical compounds used in perfumery.

EPFL2012

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system to recognize and discriminate a nearly unlimited number of structurally diverse odorant molecules. The molecular basis of OR-mediated odor signal detection and transduction is poorly understood. This is due in part to difficulties in functional expression of ORs at high yield, preventing structural and biophysical studies at the level of the receptor protein. Here we report on the heterologous expression of the mouse receptor mOR256-17 yielding an average of 106 ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression we employed different fluorescent tags. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and posttranslational fluorescence labeling of a 12 amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. The establishment of a mOR256-17-inducible HEK293S-TetO cell line allowed us to determine by fluorescence spectrofluorometry and fluorescence correlation spectroscopy (FCS) the optimal conditions to solubilize the receptor. By screening a large odorant compound library we discovered a new selective spectrum of potent mOR256-17 specific agonists. Furthermore, by using a reporter assay that couples both Gs and Gq proteins after inhibition of adenylate cyclase by the chemical adenylyl cyclase inhibitors 9-(tetrahydro-2-furanyl)-9H-purin-6amine (SQ22536) and after the inhibition of phospholipase C by the chemical inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), we identified one agonist which induced the inositol 1,4,5-trisphosphate (IP3) transduction pathway. We provided further evidence of the involvement of this second messenger (i) by employing another reporter gene specific to the Gq pathway and (ii) by using electrical impedance spectroscopy. Our attempt to create a FRET fluorophore pair system based on fluorescein arsenical hairpin (FlAsH) labeling and on carrier protein tags to study the receptor conformational changes during activation revealed that modifications in mOR256-17 sequences perturbs the correct receptor folding and prohibits its expression at the plasma membrane. Our study indicated that an essential prerequisite for achieving cell surface expression of mOR256-17 in heterologous cells is the co-expression of the accessory protein RTP1 or its short version RTP1S, which are also expressed by olfactory sensory neurons. Elsewhere, Saito and co-workers have proposed that this protein interacts with OR when the OR is expressed heterologously, but they have not provided evidence to prove the role of RTP1 towards OR. To investigate whether direct physical interactions between mOR256-17 and RTP1 occurs during receptor trafficking to the cell membrane, we labeled mOR256-17 and RTP1 orthogonally using the acyl carrier protein (ACP) and the peptidyl carrier protein (PCP) respectively. In addition, cyan fluorescent protein fused to the N-terminus of RTP1 enabled to study RTP1 biosynthesis in presence and absence of the mOR256-17; here we observed that a large portion of RTP1 remains in the endoplasmic reticulum while the mOR256-17 reaches the Golgi apparatus when it is co-expressed with RTP1S. Furthermore, by using two different RTP1S chimera, we could show that the cytosolic part of RTP1S is important for the mOR256-17 folding and trafficking.

EPFL2013

Probing Odorant-Mediated Receptor Activation

Olivia Baud

EPFL2012

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

EPFL2013