Identification of Specific Ligands for Orphan Olfactory Receptors

Abstract

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein ␣olf and adenylyl cyclase type-III to the Ca 2؉ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of G␣olf in odorant-specific signaling out of OR. The employment of the non-typical G protein ␣15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescenceimaging plate reader experiments, resulting in the deorphaning of two new OR for the odorant (؊)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (؊)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of G␣15 or G␣olf. We finally established an EC 50 -ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.

The Molecular Basis for Ligand Specificity in a Mouse Olfactory Receptor

“…This prediction was confirmed in our functional assay. The existence of antagonists for ORs has been shown in a few studies (47,48). Our results with the C12 dicarboxylic acid provide further insight into the role of OR antagonism.…”

Electronic Nose: Current Status and Future Trends

“…Copyright 2001 Elsevier. promise to increase the sensitivity and importantly selectivity. [260][261][262][263] Moreover, considering the electronic nose as a whole system, there are other possibilities to reach both of these aims. On one hand, the increase in sensitivity can be realized by appropriate sample pretreatment and preconcentration techniques, whereras filters and separation units can be used to increase the selectivity and reduce interfering substances.…”

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

“…Selection of epitope tags-The inclusion of the first 20 amino acids or the first 39 amino acids of rhodopsin (Rho-tag) at the N-terminal end has been shown to promote the cell-surface expression of some ORs 18,26,29,30 . Although we routinely use the first 20 amino acid of human rhodopsin for enhanced cell-surface expression and function, we have shown that untagged ORs can also be functionally expressed at lower efficiencies 18 .…”