Capa GPCR and NMUR in Fluid Homeostasis Figure 3. Capa, Neuromedin and Hugin receptor (CG8795)-associated calcium signatures. (A) Typical cytoplasmic Ca2+ response in S2 cells expressing capaR and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 1027 M. (B) Typical cytoplasmic Ca2+ response in S2 cells expressing NMUR 2 and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 1027 M. (C) Typical cytoplasmic Ca2+ response in S2 cells expressing CG8795and apoaequorin challenged with Drm-capa-1, Drm-PK- 1, hugg, Drm-PK-2 and NMU-25 at a concentration of 1027 M. (D) Human NMU-25 dose-response curve in S2 cells and intact tubule. NMU-25 peptide stimulation of NMUR 2- or capaR- and apoaequorin-co-transfected S2 cells; and of tubule principal cells expressing apoaequorin transgene. Cells or tubules were challenged with increasing concentrations of agonist, and [Ca2+]i was measured. Values were expressed as maximal (nM) - background (nM) (mean 6 S.E.M., N = 3). doi:10.1371/journal.pone.0029897.g003 mammalian cells [35]. Video S1 demonstrates the translocation of cytosolic– localized mammalian b-arrestin2-eYFP to the mem- brane in response to capa-1 in capaR-transfected S2 cells. To further demonstrate the direct capaR-b-arrestin-2 interaction, Bioluminescence Resonance Energy Transfer (BRET)-based b- arrestin-2 interaction assays [36] were performed by co-transfect- ing S2 cells with capaR-C-terminally tagged with Renilla luciferase and b-arrestin-2-eYFP. Capa-1 produced a clear concentration- dependent increase in BRET, reflecting capaR-b-arrestin-2 interactions, with EC50 = 8.5 mM (Fig. 2G). Agonist-induced activation of GPCRs plays a major role in the regulation of signal transduction pathways, either by propagating or terminating signals. Our data support the hypothesis that capa- 1 release regulates capaR activation and subsequent internaliza- tion, thereby also contributing to signal termination (unless the internalized receptor continues signalling). Can the physiological relevance of capaR activation and desensitization be explained by the function of the peptides? The Drosophila capa peptides have been shown to increase fluid secretion by the Malpighian tubules. However, insects generally need to retain water as much as PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29897