Investigating the molecular basis of ligand binding to and activation of olfactory receptors in living cells

Paulina Izewska
EPFL, 2006
Thèse EPFL

Résumé

The olfactory system is a highly specialized chemodetector capable of discriminating between thousands of volatile chemical substances. This enormous capacity of chemical recognition is based on a large family of olfactory receptors (ORs). Despite the fact that quite a number of putative ORs have been cloned, only limited progress has been made in functional expression of these receptors in heterologous cells. In order to functionally express odorant receptors we have established a suitable expression system in HEK 293 cells. The co-expression of the Gαq protein together with odorant receptors allowed the study of OR activation by calcium imaging. To analyze the structural properties of odorant molecules essential for OR5 and hOR17-4 binding and activation, we developed an efficient functional assay based on optical read-out of odorant responses by intracellular calcium imaging on living cells. Investigation of the ligand specificity of the two ORs derived from different species, but exhibiting significant homology on amino acid sequence level, showed a high degree of similarity in their responsive odorant molecule spectrum. A comparison of the molecular properties of odorant compounds crucial for OR5 and hOR17-4 binding and activation revealed a classification in two different structural groups. The first one comprises aromatic hydrocarbons with variable structures and sizes and the second group consists of aliphatic odorant molecules which are mainly aldehydes with variable carbon chain lengths. To elucidate further the function of ORs, the computational modeling to predict the binding site of OR5 and the docking of the small, hydrophobic odorant molecule, safrole were determined. Our findings show that the safrole binding site in OR5 involves the helical domains TM3, TM5 and TM6, which is consistent with earlier computational studies on rat I7 and mouse OR-EG receptors. The key amino acid residues appear to be hydrophobic and the van der Waals forces are implicated in safrole binding to the OR5 receptor. The mutations of the amino acid residues predicted by safrole docking analyses, Val202, Phe251 and Leu258 to Ala, indicate that these residues play an important role in OR5 activation.

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https://infoscience.epfl.ch/record/89655?ln=fr

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Paulina Izewska
EPFL, 2006
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/89655?ln=fr

À propos de ce résultat

Concepts associés (19)

Concepts associés

Chargement

Publications associées (12)

Publications associées

Chargement

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

Chargement

Characterizing molecular recognition principles of specific odorant molecule interactions with an olfactory receptor and a nuclear hormone receptor

Sylvain Etter

Olfaction, the detection of odorous chemicals in the environment, is one of the oldest mammalian sensory systems and involves large number (up to 1000) of distinct G protein coupled olfactory receptors (OR). The chemical interaction of volatile molecules with specific ORs marks the primary step in odor perception and is of critical importance for elucidating the molecular basis of detecting and discriminating thousands of odorous chemicals. Despite the fact that a number of putative ORs have been cloned, only limited progress has been made in assigning odorant molecules to specific receptors. Furthermore, the analysis of OR–ligand interactions at a cellular level is complicated by combinatorial recognition principles and thus requires large scale odorant and receptor screening to establish receptor-specific ligand profiles. Described herein are efforts towards the development of reliable and efficient assays for detecting and quantifying the responses of heterologously expressed ORs to odorants. Three strategies were employed for the optical or electrophysiological detection of ORs responses with functional assays in HEK293 cells. In two approaches, OR activation was monitored via the endogenous cAMP pathway, which was coupled either to the cAMP response element-mediated expression of different reporter genes, or to a cyclic nucleotide gated ion channel. In a third approach, OR activation was measured using Ca2+ imaging in the presence of a promiscuous Gαq protein. ORs of different species were then functionally analyzed using the different assays. A library of 250 odorant compounds representative of the chemical functional groups relevant in perfumery was screened; 16 novel specific ligands for the mouse eugenol odorant receptor (EGOR) were identified. Ten of these compounds are structurally unrelated to previously identified ligands and thus provide an extended molecular basis for describing the ligand-specificity and selectivity of EGOR in new detail. Interestingly, some ORs exhibited odorant responses in the cAMP-based assays, but not in Ca2+ imaging assays and vice-versa, which indicates a possible preferential OR coupling to specific G proteins. Such preferential coupling might be dependent on the cell type or influenced by specific odorant molecules and could potentially increase the complexity in odorantmediated signaling. A second part of this thesis describes investigations into other potential activities of odorants apart from their role in olfaction. An odorant compound library was screened to determine possible xenoestrogenic activity on human ERα expressed in cultured mammalian cells. An image-based assay to observe the nuclear redistribution processes of a functional YFP-ERα fluorescent chimera allowed the identification of seven odorant molecules that interact with the receptor. As the optical detection of YFP-ERα redistribution processes did not distinguish between agonistic and antagonistic effects, we tested the active substances in a reporter assay using luciferase under transcription control of two estrogen response elements (ERE). Two active odorants identified in the screening exhibited agonist activity as indicated by a dose-dependent activation of ERα mediated transcription of the luciferase reporter gene.

EPFL2007

Chargement

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Ligand-gated ion channels of the Cys loop family are receptors for small amine-contg. neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiol., radioligand binding, fluorescence measurements, and immunochem. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model. [on SciFinder (R)]

2003

Chargement

Characterizing molecular recognition principles of specific odorant molecule interactions with an olfactory receptor and a nuclear hormone receptor

Sylvain Etter

EPFL2007

EPFL2012