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. 2013 Jul 11;9(7):e1003619. doi: 10.1371/journal.pgen.1003619

Figure 4. RGS proteins are targets of EGL-4.

Figure 4

(A) Animals lacking each of the 8 neuronally expressed RGS proteins were tested for response to 1 mM quinine. rgs-2(lof) and rgs-3(lof) animals respond better than wild-type animals to dilute (1 mM) quinine (p<0.001). (B) RNAi knock-down of rgs-2 or rgs-3 in the quinine-detecting ASH sensory neurons of otherwise wild-type animals, using the osm-10 promoter [3], resulted in behavioral hypersensitivity to dilute (1 mM) quinine, similar to rgs-2(lof) and rgs-3(lof) animals, respectively (p>0.05 for both transgenes when compared to the respective rgs loss-of-function animals). The srb-6 promoter [61] was also used for ASH knock-down of rgs-3, and similarly resulted in hypersensitivity to 1 mM quinine (data not shown). (C) Wild-type animals overexpressing ectopic rgs-2 or rgs-3 cDNA displayed diminished response to 10 mM quinine (p<0.0001 when compared to wild-type animals). (D) rgs-2(lof);egl-4(lof) and rgs-3(lof);egl-4(lof) double mutant animals responded to dilute (1 mM) quinine similarly to egl-4(lof) animals (p>0.1 for each). (E) egl-4(gof) animals lacking either RGS-2 or RGS-3 function responded to 10 mM quinine similarly to the rgs-2(lof) and rgs-3(lof) animals, respectively (p>0.5) and (F) were hypersensitive to 1 mM quinine (p<0.001 when compared to wild-type animals). (G) rgs-2(lof) and rgs-3(lof) animals are hypersensitive to dilute (1 mM) quinine. ASH expression of wild-type RGS-2 or RGS-3, using the osm-10 promoter [3], rescued quinine hypersensitivity in the respective loss-of-function animals. The predicted PKG phosphorylation target site in each was mutated (ΔP), making RGS-2(S126A) and RGS-3(S154A). rgs-2(lof) animals expressing RGS-2(ΔP) and rgs-3(lof) animals expressing RGS-3(ΔP) remained hypersensitive to dilute quinine (p>0.05 for each). The percentage of animals responding is shown. The combined data of ≥3 independent lines, n≥120 transgenic animals, is shown. Error bars represent the standard error of the mean (SEM). Alleles used: egl-4(n479), rgs-1(nr2017), rgs-2(vs17), rgs-3(vs19), rgs-6(vs62), rgs-10(ok1039), rgs-10/11(vs109), egl-10(md176) and eat-16(tm761) loss-of-function and egl-4(ad450) gain-of-function. WT = the N2 wild-type strain. lof = loss-of-function. gof = gain-of-function.