Deorphanization of the pheromone receptor candidates MsexOR-1 and MsexOR-4

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Fig. 91. HEK 293 cells, heterologously expressing DmelSNMP-1, show significantly more [Ca²⁺] increases after control applications. Comparison of the percentages of active cells after application of 0.1 % DMSO (100 µl, p < 0.0001, Kruskal-Wallis test). The cells either were not transfected or transfected with MsexOrco, MsexOr-1, MsexOr-4, and DmelSnmp-1 in different combinations. For reasons of clarity only significant differences were indicated (* p < 0.05, ** p < 0.01,

*** p < 0.001; Dunn's multiple comparison test; n = number of experiments).

3.3.1.3 Deorphanization of the pheromone receptor candidates MsexOR-1

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Fig. 92. HEK 293 cells expressing MsexORCO and MsexOR-1 can respond to high concentrations of BAL with different kinetics. A-C. Cells were transiently transfected with MsexOrco and MsexOr-1. A. Normalized calcium imaging data for 26 HEK 293 cells. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. After application of 10^-9 M bombykal (BAL, 100 µl), all cells showed slow [Ca²⁺] increases with superimposed transient increases. B. BAL (100 µl, 10^-6 M) was applied before the recording started; after more than 200 s 16 of 41 cells showed transient [Ca²⁺] increases. C. The percentages of active cells after application of different concentrations of BAL or 0.1 % DMSO were compared (p=0.0006, Kruskal-Wallis test, significant differences are indicated by asterisks). D. Comparison of the percentages of active cells, transfected with MsexOrco and MsexOr-4 (p=0.649, Kruskal-Wallis test). E. The medial concentrations of BAL (10^-12 and 10^-9 M) were pooled for comparison of cells with different expression profiles (p = 0.3207, Kruskal-Wallis test). * p < 0.05, ** p < 0.01, *** p < 0.001; Dunn's multiple comparison test, n = number of experiments.

When cells transfected with MsexOrco and MsexOr-1 were stimulated with different concentrations of bombykal, only the highest concentration (10^-6 M) resulted in a significantly higher percentage of active cells (median: 6.3 %) compared to the lower concentrations and the solvent control (0.1 % DMSO). This suggests that MsexOR-1 might be the bombykal receptor (Fig. 92). However, since micromolar concentration exceed the physiological range, the experiments were focused on lower concentrations. Cells transfected with MsexOrco and MsexOr-4 were only stimulated with 10^-12 and 10^-9 M bombykal. As shown for cells transfected with MsexOrco and MsexOr-1, the percentages of active cells after application of bombykal in these concentrations did not differ from each other or

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control applications. Henceforth experiments employing these concentrations were pooled. The proportion of responding cells, transfected with MsexOrco and MsexOr-1/4 was not different from control cells, either non-transfected or transfected with MsexOrco or MsexOr-1 alone (Fig. 92).

In the olfactory system of mice Ca²⁺-calmodulin (Ca²⁺-CaM)-modulation plays a key role in ion channel desensitization and thus response termination and adaptation (Song et al. 2008; Spehr et al.

2009). Therefore experiments were performed to investigate whether the low bombykal-sensitivity could be due to Ca²⁺-CaM modulation of the signaling components. Cells transfected with MsexOrco and MsexOr-1 (n = 4 experiments) or non-transfected cells (n = 5) were incubated for 10 min with the CaM antagonist W7 before bombykal stimulation (Fig. 93). However, in this experimental series almost no active cells were detected, neither in control experiments before W7 incubation, nor during W7 incubation, or after bombykal stimulation.

Fig. 93. The bombykal-sensitivity is not affected by calmodulin inhibition. Percentages of active cells after subsequent application of DMSO, the calmodulin inhibitor W7 (50 µM, 100 µl), and bombykal (BAL, 10^-12 or 10^-9 M, 100 µl) are shown. The transfection profile is indicated in the legend. Each symbol represents one experiment.

The low bombykal responsiveness of the cells could have several reasons, including the possibility that MsexORCO did not function as (part of) an ion channel. To test this hypothesis, one can benefit from the fact, that ORCO orthologues of different insect species share very high sequence similarities and may substitute for each other (Jones et al. 2005). First, it was tested, whether a substitution of MsexORCO by DmelORCO might improve the bombykal sensitivity. Therefore, HEK 293 cells were transiently transfected with DmelOrco and MsexOr-1 and stimulated with different bombykal concentrations (n = 3 experiments). Due to the low number of experiments no statistical analysis was performed, but apparently the percentages of active cells did not differ between different bombykal concentrations and the solvent control (Fig. 94, Tab. 30, Tab. 31).

Fig. 94. Replacement of MsexORCO by DmelORCO does not appears to improve the bombykal sensitivity. The percentages of active cells after application of bombykal in different concentrations or 0.1 % DMSO are shown. Each symbol represents the percentage of active cells of one experiment. The HEK 293 cells were transfected with DmelOrco and MsexOr-1. Each symbol represents one experiment.

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Next, MsexOR-1 or MsexOR-4 was heterologously expressed in an insect cell line. For this purpose SF9 cells were chosen, which previously were shown to express endogenous ORCO (SfruORCO, Smart et al. 2008). Thus, SF9 cells were transiently transfected with one of the male specific MsexOrs and stimulated with 10^-12 or 10^-9 M bombykal. In some experiments [Ca²⁺] increases after bombykal application were detected, but the proportion of responding cells was neither different between MsexOr-1- and MsexOr-4-transfected cells nor between bombykal and control applications (Fig. 95, Tab. 30, Tab. 31). The expression of endogenous SfruORCO in the SF9 cells was not verified in this thesis. Since DmelORCO was shown to be activated by cAMP, control experiments were performed to test the SF9 cells for cAMP sensitivity, which could hint at a possible ORCO expression. In these experiments slightly more cells responded to the adenylyl cyclase activator forskolin (10^-5 M, n = 10), compared to the solvent control, although not significant (Fig. 127, Tab. 30, Tab. 31).

Fig. 95. SF9 cells, transiently transfected with MsexOr-1 or MsexOr-4, do not reliably respond to bombykal. A. Normalized calcium imaging data for 115 SF9 cells, transfected with MsexOr-1. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. After application of 10^-12 M bombykal (BAL, 100 µl, arrow), three cells showed threshold-exceeding [Ca²⁺] increases with different kinetics. B. The cells were transfected with MsexOr-4. After application of 10^-12 M bombykal three of 124 cells showed threshold-exceeding [Ca²⁺] increases. C. The percentages of active cells after application of bombykal (10^-12 or 10^-9 M) or 0.1 % DMSO were compared (n.s. = not significant, Mann-Whitney test, n = number of experiments). The transfection profile of the cells is indicated in the legend.

Another option to exclude MsexORCO as factor of uncertainty was the heterologous coexpression of the pheromone receptor candidates together with the murine G protein α-subunit Gα15, which was shown to couple various receptors to the IP3 signaling cascade (Offermanns and Simon 1995). Similar to the experiments described above, HEK 293 cells, transiently transfected with gα15 and MsexOr-1 (n = 6) or MsexOr-4 (n = 9) did not reliably respond to bombykal at concentrations of 10^-12 or 10^-9 M.

For each MsexOr-1 or MsexOr-4 some cells showing [Ca²⁺] increases were detected, but the percentage of cells was neither different from each other nor from control experiments (Fig. 96, Tab. 30, Tab. 31). Thus, the low responsiveness to bombykal could neither be improved by replacement of MsexORCO with DmelORCO or SfruORCO nor by expression of the ORCO-independent Gα15 signaling system.

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Fig. 96. Heterologous expression of the murine G protein subunit Gα15 does not improve bombykal-sensitivity. A. Normalized calcium imaging data for 40 HEK 293 cells, transfected with gα15 and MsexOr-1. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. After application of 10^-9 M bombykal (BAL, 100 µl, arrow), six cells showed [Ca²⁺] increases with different kinetics.

B. The cells were transfected with gα15 and MsexOr-4. After application of 10^-9 M bombykal one of 105 cells showed a long-lasting [Ca²⁺] increase. C. The percentages of active cells after application of bombykal (10^-12 or 10^-9 M) or 0.1 % DMSO were compared (n.s. = not significant, Mann-Whitney test, n = number of experiments). The transfection profile of the cells is indicated in the legend.

Another possibility for the low responsiveness could be that other crucial parts of the signaling system, required for pheromone responses, were missing in the heterologous expression systems.

One of those signaling components could be SNMP-1, which is expressed in the pheromone-sensitive ORNs of M. sexta and other insect species, and is involved in pheromone responses via unknown mechanisms (Rogers et al. 2001a; Vogt et al. 2009; Li et al. 2014b; Pregitzer et al. 2014). Therefore, the effects of SNMP-1 coexpression were examined (Fig. 97, Tab. 30, Tab. 31). First, the D. melanogaster orthologue of snmp-1 (DmelSnmp-1) was used for cotransfection of HEK 293 cells.

Cells, transfected with MsexOrco, MsexOr-1, and DmelSnmp-1, showed more [Ca²⁺] increases (n = 21, median: 6.1 %) after bombykal application (10^-12 and 10^-9 M) than cells, transfected with MsexOrco, MsexOr-4, and DmelSnmp-1 (n = 17, median: 1.75 %). However, both groups lacked significant differences with the control groups, which were stimulated with the solvent control. Next, the M. sexta snmp-1 orthologue (MsexSnmp-1) was cotransfected with MsexOrco and MsexOr-1 or MsexOr-4, but the dedicated experiments did not show any significant bombykal responses, neither for those experiments employing bombykal, dissolved in fatty acid-free BSA (compared with the solvent control), nor for those experiments performed in bath solution containing DMSO or fatty acid-free BSA (MsexOr-1 and MsexOr-4 compared with each other or spontaneous [Ca²⁺] increases, Fig. 97).

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Fig. 97. Coexpression of SNMP-1 does not improve the bombykal-sensitivity. A, B. Percentages of active cells after application of bombykal (BAL, 10^-12 and 10^-9 M) or the respective control solution containing 0.1 % DMSO (A) or 10^-6 - 10^-4 M bovine serum albumin (BSA, B). The experiments were performed in standard ringer solution. C, D. The experiments were performed in bath solution containing 0.1 % DMSO (C) and 10^-6 - 10^-4 M BSA (D) respectively. Instead of applying control solution, spontaneous [Ca²⁺] increases were monitored. HEK 293 cells were transfected with different combinations of MsexOrco, MsexOr-1, MsexOr-4, MsexSnmp-1 (MsSnmp), or DmelSnmp-1 (DmSnmp). The transfection profile of the respective cells is shown in the legends. All data are shown as box plots with whiskers (from minimum to maximum). Data groups were compared using the Kruskal-Wallis test (comparison of the percentages of active cells, transfected with MsexOrco, MsexOr-1, and MsexSnmp-1 in C, p=0.7147) or Mann-Whitney test (all other comparisons, A-D). Significant differences are indicated by asterisks (n.s. = not significant, ** p < 0.01, n = number of experiments).

Remarkably, in all experiments performed with cells expressing MsexSNMP-1 or DmelSNMP-1, the percentage of active cells was slightly higher than in experiments performed without SNMP-1. For example, the average percentage of active cells, transfected with MsexOrco and MsexOr-1 after bombykal stimulation (10^-12 and 10^-9 M) was 0.0 % (Fig. 92 E), while cotransfection of DmelSnmp-1 increased the percentage to 6.1 % (Fig. 97 A, Tab. 30, Tab. 31). However, as shown before (Fig. 90, Fig. 91) the cotransfection of snmp-1 also increased the percentage of active cells after control applications as well as the percentage of cells showing spontaneous [Ca²⁺] increases, explaining the lack of significant differences in comparisons with the control experiments. Thus, the coexpression of MsexSNMP-1 or DmelSNMP-1 failed to improve the bombykal responsiveness, but instead elevated the "general activity and responsiveness" of the cells.

Next to bombykal, the main compound of M. sexta's pheromone blend, one of the minor compounds, (E,E,Z)-10,12,14-hexadecatrienal, is required for the characteristic, behavioral pheromone response of the male (Tumlinson et al. 1989). The effect of this compound can be

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simulated by (E,Z)-11,13-pentadecadienal (C-15), which is chemically more stable (Christensen and Hildebrand 1987). In the following experiments it was tested, whether C-15 might be detected by one of the male-specific ORs. Thus, HEK 293 cells, transfected with MsexOrco, MsexOr-1/4, and MsexSnmp-1 were stimulated with low concentrations of C-15 (20*10^-15 - 10^-12 M). As shown for the bombykal stimulations almost no responding cells were found, and the rare [Ca²⁺] increases varied in their kinetics (Fig. 98, Tab. 30, Tab. 31). Again, the experiments were performed under different conditions, regarding bath solution and solvent of C-15. While no significant differences were detected between C-15 stimulation and control experiments for cells expressing MsexOR-1, the percentage of responding cells, expressing MsexOR-4, (median: 6.58 %) was significantly higher compared to spontaneous [Ca²⁺] increases (median: 1.0 %) in experiments, performed in the continuous presence of 0.1 % DMSO in the bath (Fig. 98).

In conclusion, it was not possible to deorphanize the pheromone receptor candidates MsexOR-1 and MsexOR-4. The present experiments hint, that MsexOR-1 could be the receptor, detecting bombykal (Fig. 92, Fig. 97), and MsexOR-4 could be the receptor, detecting (E,E,Z)-10,12,14-hexadecatrienal (Fig. 98), but an unequivocal identification was not accomplished.

Fig. 98. Cells, heterologously expressing MsexOR-4, can respond to C-15. A. Normalized calcium imaging data for 76 HEK 293 cells transfected with MsexOrco, MsexOr-4, and MsexSnmp-1. Each line represents the percentage deviation of the fluorescence ratio from the mean of the first ten values (% Δ(F340/F380)) for one cell. After application of (E,Z)-11,13-pentadecadienal (C-15, 100 µl, 20 pM, arrow), five cells showed threshold-exceeding [Ca²⁺] increases with different kinetics and delays. B. Comparison of the percentages of active cells after application of C-15 (20 fM - 20 pM) or fatty acid-free bovine serum albumin (BSA(-), 10^-5 - 10^-6 M) and the percentages of cells showing spontaneous [Ca²⁺] increases respectively (n.s. = not significant, * p < 0.05, Mann-Whitney test, n = number of experiments). The transfection profile of the cells is indicated in the legend and the bath solution of the underlying experiments at the bottom.

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