Capa GPCR and NMUR in Fluid Homeostasis medium to allow resensitization. Nuclei are labelled blue with DAPI, scale bar represents 10 mM. (D) S2 cells expressing capaR were left untreated (0), or treated for 5, 10, 20 or 30 minutes (indicated) with 1027 M capa-1 to induce receptor internalization. An additional sample was incubated for 30 minutes with capa-1, washed three times with culture medium without capa-1 followed by 30 minutes incubation in culture medium to allow resensitization (Res.). A sample of untransfected cells serves as a negative control. Samples were subjected to cell surface biotinylation to label plasma membrane proteins. We found that the protein concentration of biotinylated samples are generally lower than that of the total lysates; therefore, the equivalent of 5000 cells were loaded for the total lysate, and an equivalent of 15,000 cells were loaded for the biotinylated samples. Total lysates and biotinylated samples were subjected to western blot analysis. Immunoblot using anti-capaR antibody identified a band of the predicted size of 52 kDa which confirms the specificity of the antibody and an additional non specific 75 kDa protein absent in the cell-surface (biotinylated) fraction. (E) Samples from the cell surface biotinylation experiment were semi-quantified and corrected for total receptor expression. Relative cell surface expression is shown as a percentage of the non-treated S2 cells expressing capaR (t = 0). Bars indicated with an asterisk were significantly (P,0.05 as determined by one-way ANOVA) reduced compared to t = 0. (F) Calcium measurements in S2 cells transfected with expression constructs for aequorin and the capa receptor. S2 cells were challenged with 1027 M capa-1, pre-treated with capa-1 for 15 min (Desensitization (Des.)), followed by ligand removal after which S2 cells were challenged at 15 min or 30 min (Resensitization (Res.)) with 1027 M capa-1 and cytosolic [Ca2+]i levels measured. Bars indicated with an asterisk were significantly (P,0.05 as determined by Student’s t-test) reduced compared to control. (G) Analysis of capaR-b-arrestin-2 interactions. S2 cells were co-transfected with capa receptor tagged with Renilla luciferase and b-arrestin-2 tagged with eYFP. Bioluminescence Resonance Energy Transfer (BRET) signals were monitored after treatment of the cells for 15 min with varying concentrations of capa-1. Data are expressed as mBRET units 6 SEM, N = 3. doi:10.1371/journal.pone.0029897.g002 Statistical analysis Data are presented as mean 6 S.E.M. Significance of differences was assessed with Student’s t-test (two-tailed) for unpaired samples or one-way ANOVA, with significance taken as P,0.05, marked graphically with an asterisk. Results and Discussion Capa receptor-associated calcium signatures In Drosophila S2 cells assays, CG14575 encodes a functional receptor for both the capa-1 and capa-2 peptides [11,12] (Fig. 1A). Stimulation of capaR with both capa-1 and capa-2 results in a biphasic rise in [Ca2+]i, comprising a rapid primary peak followed by a slower secondary peak. The secondary [Ca2+]i response is abolished when external Ca2+ is removed (Fig. 1B). CG14575 responds to both capa-1 and capa-2 in a dose- dependent manner (Fig. 1C). EC50 values for stimulated [Ca2+]i responses for capa-1 and capa-2 in the nM range (3.06 nM, 4.32 nM respectively). CG14575-encoded receptor also responds to the lepidopteran peptide CAP2b [28], a member of the capa family (data not shown). Previous work on CG14575 - transfected CHO cells [11] and Xenopus oocytes [12] show values in the 1027 M range. Here we show Capa-induced [Ca2+]i increases are similar in size and dynamics to the response seen in principal cells of intact Malpighian tubules [3], (Table S1), with EC50 values at nM concentration. In intact tubules, removal of external calcium, or pharmaco- logical/genetic intervention of plasma membrane channels including L-type, CNG and TRP/TRPL channels significantly reduce or even abolish CAP2b/capa-stimulated [Ca2+]i responses Table 1. Peptide Human NMU-25 Porcine NMU-8 Drm-capa-1 Drm-capa-2 Aplysia SCPB Drm-PK-2 hugg Sequence FRVDEEFQSPFASQSRGYFLFRPRN-amide YFLFRPRN-amide GANMGLYAFPRV-amide ASGLVAFPRV-amide MNYLAFPRM-amide SVPFKPRL-amide pQLQSNGEPAYRVRTPRL-amide Drm-PK-1 (capa-3) TGPSASSGLWFGPRL-amide doi:10.1371/journal.pone.0029897.t001 [29]. The primary [Ca2+]i response is due to release from internal Ca2+ stores (Fig. 1B) via the Phospholipase C (PLC)/Ins 1,4,5 trisphosphate (InsP3) pathway [30] (Fig. 1D), where the capa-1- induced [Ca2+]i response is inhibited by the widely-used PLC inhibitor (U73122) in a dose-dependent manner. Overall, these S2 cell data are consistent with the role of capa- sensitive intracellular calcium channels and of the role of influx of extracellular calcium via plasma membrane calcium channels in the tubule. Desensitization and internalization of the capaR GPCRs undergo desensitization and/or internalization shortly after agonist stimulation. eYFP-tagged capaR-transfected S2 cells were used to address the internalization/recycling process for capaR (Fig. 2A–C). Capa-1 treatment results in internalization (intracellular vesicles) (Fig. 2B) after which endocytic recycling to the plama membrane occurs during resensitization in the absence of capa-1 (Fig. 2C). Cell-surface biotinylation experiments can reliably indicate the status of receptors and membrane-associated proteins as this technique permits the labeling and purification of plasma membrane proteins [31]. In resting capaR transfected S2 cells, the capaR signal is very strong in the biotinylated fraction indicating high level of capaR at the plasma membrane, but upon stimulation with 1027 M capa-1, the cell-surface (biotinylated) fraction was reduced and quickly restored upon removal of the agonist (Fig. 2D). Quantification of plasma membrane-localized capaR (Fig. 2E) showed that the capaR signal is significantly reduced during desensitization, due to internalization, and then recycled to the plama membrane during resensitization. To what extent is capaR signalling affected during internaliza- tion/recycling? [Ca2+]i measurements showed that pre-treatment of S2-capaR cells with 1027 M capa-1 for 15 min completely abolished the capa-1 induced calcium response, indicating desensitization of the capaR (Fig. 2F). After ligand removal, S2 cells were challenged at 15 min or 30 min with 1027 M capa-1 and cytoslic [Ca2+]i levels measured. A 15-minute resensitization period sees a modest capa-1 [Ca2+]i response and a response similar to control at 30 min (Fig. 2F), correlating with the % of plasma membrane-localized capaR (Fig. 2E). GPCRs are desensitized by a GPCR Regulating Kinase (GRK)/arrestin-mediated mechanism. In Drosophila, two GRKs [32] and a non-visual arrestin [33] have been identified. Desensitization of capaR involves a GRK-associated mechanism, as capa-1 induces increased GRK-2 gene expression in tubules (Fig. S2). Desensitization of insect [34] and mammalian GPCRs can also be assessed by translocation of b-arrestin in transfected PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29897