Figure 1.
Logic of RNAi-based deorphanization experiment.
The general set of experimental outcomes for an RNAi-based deorphanization experiment focused on the G and G
pathway are shown. Letters
and
each represent cAMP datasets for particular treatment conditions. Potential results are described with respect to the notion that a given ligand may act on multiple GPCRs that are not necessarily coupled to the same G protein (G
or G
). Abbreviations: NT, no treatment; Fk, forskolin; L, ligand; R, receptor; RNAi-control, control membrane preparation; RNAi-R, R-suppressed membrane preparation;
, cAMP measurement variable. Asterisks (*) are used to denote normalized data.
Figure 2.
Peptide and biogenic amine ligand cAMP screen performed against isolated D. tigrina membranes.
RIA cAMP outputs are normalized and shown as mean SEM, with asterisks representing statistically significant differences compared to a control bar (
); *P
0.05, **P
0.01, ***P
0.001, one-way ANOVA, Tukey post hoc test. Top: red-outlined bars signify ligands that stimulate cAMP compared to basal levels, likely mediated by G
-coupled GPCRs. Bottom: red-outlined bars signify ligand inhibition of Fk-stimulated cAMP, likely mediated by G
-coupled GPCRs. Serotonin (5-HT) stimulates basal cAMP, while octopamine (Oct), GYRIFamide (GYIRF), and neuropeptide F (NPF) all inhibit Fk-stimulated cAMP at 100 uM. These changes in cAMP are presumably receptor-mediated, and should therefore be altered in a ligand-specific manner by subtraction of particular receptor targets from cell membranes via RNAi.
Figure 3.
Semi-quantitative PCR reveals GtNPR-1 knockdown.
Lane 1 is a 100 bp DNA ladder, lanes 2–5 represent individual GtNPR-1 dsRNA-fed planarians, and lanes 6–9 represent control dsRNA-fed planarians. The bottom band (∼300 bp) is the 18S internal standard, and the top band (∼400 bp) shows GtNPR-1 expression. The top band disappears in the experimental group, confirming near abolishment of receptor expression in these worms. Relative band intensities (GtNPR-1/18S rRNA) for GtNPR-1 RNAi group: 0.440.15. Relative band intensities for control group (band location manually selected): 0.08
0.02. This corresponds to
80% knockdown of GtNPR-1 transcript.
Figure 4.
RNAi-based GtNPR-1 deorphanization.
Treatment groups are Control (control dsRNA) and GtNPR-1 RNAi (GtNPR-1 dsRNA). Treatments are C (control), Fk (10 M forskolin), and Fk
GYIRF (10
M forskolin and 10
M GYIRFamide). Each bar is the mean (
SEM) of 3 individual experiments. Basal cAMP levels were set as a baseline for each individual experiment, and cAMP values were normalized with respect to the level of Fk-stimulated cAMP (set at 100%). This allowed us to join datasets with differing basal cAMP levels, due to variance in the quality and yield of individual membrane preparations. Analysis of the raw cAMP values of individual experiments renders the same results (Table 1). Asterisks indicate significance at P
0.001 (***), and “ns” indicates no significant difference (one-way ANOVA, Tukey post hoc test).
Table 1.
RNAi-based GtNPR deorphanization cAMP raw values.
Table 2.
5-HT receptor candidate selection.
Figure 5.
Maximum parsimony tree of serotonin receptors.
Phylogenetic analysis was performed using planarian (S. mediterranea and D. japonica), parasite (S. mansoni), human and C. elegans 5-HT receptors and putative 5-HT receptors. TM domains I-VII were were extracted from the alignment for bootstrapping (bootstrap value 1000). Outlined receptors are significantly diverged from vertebrate and ecdysozoan serotonin receptors. DtSER-1 (red) was amplified using a degenerate PCR strategy and was chosen to undergo RNAi-based deorphanization.
Figure 6.
Multiple sequence alignment of serotonin receptors.
DtSER-1 is shown aligned with other putative flatworm 5-HT receptor sequences that fall into to the same phylogenetic grouping (highlighted in Figure 5). This analysis encompasses low-entropy TM domains I–V, and TM domains are demarcated above the alignment in blue as predicted by HMMTOP [50]. Consensus (absolutely conserved) residues are shown for the flatworm receptors, and those conserved between this flatworm receptor grouping and a human serotonin receptor (5-HT5A) are highlighted in blue.
Figure 7.
Semi-quantitative PCR reveals DtSER-1 knockdown.
Lane 1 is a 100 bp DNA ladder, lanes 2–5 represent individual DtSER-1 dsRNA-fed planarians, and lanes 6–9 represent control dsRNA-fed planarians. The bottom band (∼300 bp) is the 18S internal standard, and the top band (∼480 bp) shows DtSER-1 expression. The top band disappears in the experimental group, confirming near abolishment of DtSER-1 receptor expression in these worms.
Figure 8.
RNAi-based DtSER-1 deorphanization.
Treatment groups are Control (control dsRNA) and DtSER-1 RNAi (DtSER-1 dsRNA). Treatments are C (control) and 5-HT (10 M). Each bar is the mean (
SEM) of 3 individual experiments. cAMP levels were normalized to basal cAMP levels (set at 100%), and the datasets were joined. DtSER-1 knockdown corresponds to significantly decreased cAMP stimulation (∼32%) in response to 5-HT. Analysis of the raw cAMP values of individual experiments renders the same results (Table 3). Asterisks indicate significance at P
0.001 (***), and “ns” indicates no significant difference (one-way ANOVA, Tukey post hoc test).
Table 3.
RNAi-based DtSER-1 deorphanization cAMP raw values.