Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Insect Biochem Mol Biol
2020 Feb 01;117:103289. doi: 10.1016/j.ibmb.2019.103289.
Show Gene links
Show Anatomy links
Functional characterization of odorant receptors from the moth Eriocrania semipurpurella: A comparison of results in the Xenopus oocyte and HEK cell systems.
Hou X
,
Zhang DD
,
Yuvaraj JK
,
Corcoran JA
,
Andersson MN
,
Löfstedt C
.
???displayArticle.abstract???
The Xenopus oocyte and the Human Embryonic Kidney (HEK) 293 cell expression systems are frequently used for functional characterization (deorphanization) of insect odorant receptors (ORs). However, the inherent characteristics of these heterologous systems differ in several aspects, which raises the question of whether the two systems provide comparable results, and how well the results correspond to the responses obtained from olfactory sensory neurons in vivo. Five candidate pheromone receptors were previously identified in the primitive moth Eriocrania semipurpurella (Esem) and their responses were characterized in HEK cells. We re-examined the responses of these five EsemORs in Xenopus oocytes. We showed that in both systems, EsemOR1 specifically responded to the plant volatile β-caryophyllene. EsemOR3 responded stronger to the pheromone component (S,Z)-6-nonen-2-ol than to its enantiomer (R,Z)-6-nonen-2-ol, the second pheromone component. However, EsemOR3 also responded secondarily to the plant volatile β-caryophyllene in the oocyte system, but not in the HEK cell system. EsemOR4 was unresponsive in the HEK cells, but responded primarily to (R,Z)-6-nonen-2-ol followed by (S,Z)-6-nonen-2-ol in the oocytes, representing a discovery of a new pheromone receptor in this species. EsemOR5 was broadly tuned in both systems, but the rank order among the most active pheromone compounds and antagonists was different. EsemOR6 showed no response to any compound in either system. We compared the results obtained in the two different heterologous systems with the activity previously recorded in vivo, and performed in situ hybridization to localize the expression of these OR genes in the antennae. In spite of similar results overall, differences in OR responses between heterologous expression systems suggest that conclusions about the function of individual ORs may differ depending on the system used for deorphanization.
Fig. 1. Responses of EsemOR4 in oocytes and HEK293 cells. (A) Representative current traces of oocytes upon successive exposures to 100 μM stimuli. Each compound was applied at the time indicated by the arrowheads for 20 s. Upper and lower traces include different sets of test stimuli. (B) Normalized responses (relative to primary ligand in each system) of EsemOR4 in the oocytes (white bars) and HEK cells (black bars). (C) Hedges’ g standardized unbiased effect size of stimuli in relation to the DMSO control of EsemOR4 in oocytes (white bars) and HEK293 cells (black bars). (D) Comparison of dose-responses of EsemOR4 in oocytes (solid line) and HEK293 cells (dashed line). Error bars are s.e.m. in (A) and (D), and 95% CI in (C). N-values for the HEK-cell data refer to independent experiments, each containing three technical replicates (Ntotal = 9).
Fig. 2. Responses of EsemOR1 in oocytes and HEK293 cells. (A) Representative current traces of oocytes upon successive exposures to 100 μM stimuli. Each compound was applied at the time indicated by the arrowheads for 20 s. Upper and lower traces include different sets of test stimuli. (B) Normalized responses (relative to primary ligand in each system) of EsemOR1 in the oocytes (white bars) and HEK cells (black bars). (C) Hedges’ g standardized unbiased effect size of stimuli in relation to the DMSO control of EsemOR1 in oocytes (white bars) and HEK293 cells (black bars). (D) Comparison of dose-responses of EsemOR1 in oocytes (solid line) and HEK293 cells (dashed line). Error bars are s.e.m. in (A) and (D), and 95% CI in (C). N-values for the HEK-cell data refer to independent experiments, each containing three technical replicates (Ntotal = 9).
Fig. 3. Responses of EsemOR3 in oocytes and HEK293 cells. (A) Representative current traces of oocytes upon successive exposures to 10 μM stimuli. Each compound was applied at the time indicated by the arrowheads for 20 s. Upper and lower traces include different sets of test stimuli. (B) Normalized responses (relative to primary ligand in each system) of EsemOR3 in the oocytes (white bars) and HEK cells (black bars). (C) Hedges’ g standardized unbiased effect size of stimuli in relation to the DMSO control of EsemOR3 in oocytes (white bars) and HEK293 cells (black bars). (D) Comparison of dose-responses of EsemOR3 in oocytes (solid line) and HEK293 cells (dashed line). Error bars are s.e.m. in (A) and (D), and 95% CI in (C). N-values for the HEK-cell data refer to independent experiments, each containing three technical replicates (Ntotal = 9).
Fig. 4. Responses of EsemOR5 in oocytes and HEK293 cells. (A) Representative current traces of oocytes upon successive exposures to 100 μM stimuli. Each compound was applied at the time indicated by the arrowheads for 20 s. Upper and lower traces include different sets of test stimuli. (B) Normalized responses (relative to primary ligand in each system) of EsemOR5 in the oocytes (white bars) and HEK cells (black bars). (C) Hedges’ g standardized unbiased effect size of stimuli in relation to the DMSO control of EsemOR5 in oocytes (white bars) and HEK293 cells (black bars). (D) Comparison of dose-responses of EsemOR5 in oocytes (solid line) and HEK293 cells (dashed line). Error bars are s.e.m. in (A) and (D), and 95% CI in (C). N-values for the HEK-cell data refer to independent experiments, each containing three technical replicates (Ntotal = 9).
Fig. 5. Expression pattern of EsemORs/Orco in adult male antennae. In situ hybridization with biotin-labelled probes for EsemORs as shown in green signal and DIG-labelled probes for EsemOrco as shown in magenta signal.
