Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

Abstract

Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs.

Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs

“…intracellular loop] and is capable to activate them independently from each other in a ligand-dependent manner. These results also support a recent published paper in which the authors showed the capability of MIEG and other known agonists to activate heterologously overexpressed Golf/Gna15 in HEK293T cells in a ligand-dependent manner (Ukhanov et al, 2014 ). As Gnao is highly expressed in juvenile mice (before Golf and other cAMP signaling components are upregulated during maturation), this pathway might reflect an odor recognition system predominantly used in embryonic and juvenile mice before cAMP signaling is fully developed.…”

“…To solve this issue, we decided to perform our experiments on Olfr73-expressing OSNs, as we could use transgenic Olfr73-GFP mice (Oka et al, 2006 ), allowing us to identify Olfr73-positive OSNs for further experiments. Furthermore, Olfr73 is one of the best characterized murine ORs, with various known strong and weak agonists (Baud et al, 2011 ), and most of the previously performed alternate olfactory signaling studies analyzed this receptor (Oka et al, 2004 ; Ukhanov et al, 2014 ).…”

“…Furthermore, there are several studies indicating an influence of DAG- and PLC-dependent alternative olfactory signaling pathways in specific subsets of OSNs (Yu et al, 2014 ) and specialized OSNs expressing Trace amine–associated receptors (TAARs; Liberles and Buck, 2006 ) or Guanylyl cyclase-D (GC-D; Juilfs et al, 1997 ) instead of the ORs. Even more complexity in signaling modes is attained because heterologously expressed ORs can activate different G proteins in a stimulus-dependent manner, leading to a duality of intracellular signaling effects (Ukhanov et al, 2014 ). Generally, a second sensory pathway might promote the system to elicit, fine-tune, regulate, or diminish odor responses.…”

Rapid Deorphanization of Human Olfactory Receptors in Yeast

“…ORs sometimes bind different ligands depending on the G α subunit to which they are coupling. 23,24 Sometimes, an OR is activated by the same ligand independent of the G α subunit to which it is coupled, and when it is coupled to G olf , a signal enhancement in observed. 25,26 Nerol was discovered as a ligand for OR2W3 using G olf .…”

Commentary: Oxygen regulation of breathing through an olfactory receptor activated by lactate

“…The paper by Chang et al ( 2015 ) has uncovered a new important transduction mechanism of CB response to hypoxia. However, since olfactory receptors are able to couple to multiple G proteins activating adenylate cyclase (AC) or phospholipase-C (PLC) pathway (Ukhanova et al, 2014 ), the signaling downstream lactate-activated Olfr78 in CB needs to be further investigated. Indeed, it can differ from that of olfactory neurons: Olfr78 activation in CB could directly induce Ca ++ transients or could lead to a K + channel closure by an indirect mechanism.…”