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.

A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level.

We have screened an odorant compound library and discovered molecules acting as chemical signals that specifically activate both G-protein-coupled olfactory receptors (ORs) on the cell surface of olfactory sensory neurons and the human nuclear estrogen receptor ␣ (ER) involved in transcriptional regulation of cellular differentiation and proliferation in a wide variety of tissues. Hence, these apparent dual active odorants induce distinct signal transduction pathways at different subcellular localizations, which affect both neuronal signaling, resulting in odor perception, and the ER-dependent transcriptional control of specific genes. We demonstrate these effects using fluorescence-based in vitro and cellular assays. Among these odorants, we have identified synthetic sandalwood compounds, an important class of molecules used in the fragrance industry. For one estrogenic odorant we have also identified the cognate OR. This prompted us to compare basic molecular recognition principles of odorants on the two structurally and apparent functionally non-related receptors using computational modeling in combination with functional assays. Faced with the increasing evidence that ORs may perform chemosensory functions in a number of tissues outside of the nasal olfactory epithelium, the unraveling of these molecular ligand-receptor interaction principles is of critical importance. In addition the evidence that certain olfactory sensory neurons naturally co-express ORs and ERs may provide a direct functional link between the olfactory and hormonal systems in humans. Our results are therefore useful for defining the structural and functional characteristics of ER-specific odorants and the role of odorant molecules in cellular processes other than olfaction.Our nose detects a large variety of odorant signals relying on about 350 different predicted G-protein-coupled olfactory receptors (ORs) 3 with a conserved seven-transmembrane helical structure (1-3). By binding odorant molecules ORs modulate the conversion of chemical into electrical neuronal signals that are finally decoded in higher brain regions to trigger emotional and behavioral responses (4 -6). Odorants are generally small, hydrophobic organic molecules with highly variable chemical structures and properties (7). They easily pass cell membranes, and, due to their enormous chemical diversity, some of them might, apart from their "conventional" role in olfaction, trigger also yet unknown cellular processes. The role of specific ORs in sperm chemotaxis has been documented (8,9), and the widespread ectopic expression of OR genes in a large number of non-olfactory human tissues implicates additional, unproven functions of ORs in embryonic development and cell-cell recognition (10), and proliferation rates in prostate cancer cells (11). Recent findings indicate that ORs might also have chemosensory functions in kidney (12). Although most present efforts concentrate on matching odorants with their cognate ORs to define their molecular receptive ranges and receptor fu...

On page 30551, Table 1, first column, the experimentally determined values of EC 50 of ER activation for MC, Polysantol, Javanol, and androstenol have been presented with the wrong concentration (mM). The correct concentration is M.

a b s t r a c tThe human estrogen receptor alpha (ERα) mediates the proliferative action of hormones in breast cancer cells by regulating the expression of target genes to control cellular functions. Current methodologies do not permit a real-time assessment of these processes in living cells. We overcome this limitation using electrical cell-substrate impedance sensing for measuring ERα-regulated signaling processes indicative of the onset of cell proliferation to target them for compound screenings. We report that hormone like odorants regulate, similarly as natural estrogen, ERα-mediated gene expression involved in mitogenic and developmental processes in MCF7 breast cancer cells. An odorant concentration-dependent switch in cell responses was detectable already 10-15 h post-stimulation, providing rapid quantification of hormonal activity before cell division occurred. Though ERα exhibits complex regulatory roles our noninvasive approach captures its activity for accelerated screenings of compounds promoting breast cancer cell proliferation expanding the analysis of ERα signaling networks.