Capa GPCR and NMUR in Fluid Homeostasis Figure 5. Immunocytochemical localization of the capa-expressing neurons with a vertebrate NMU antibody. Larval central nervous system was doubly labeled using an antibody against capa precursor (A, red), an antibody against porcine neuromedinU-8 (B, green). The immunoreactive cells bodies and neurohemal organs, the retrocerebral complex for the 2 cell bodies in the subesophageal neuromere, and the abdominal median transverse nerves for the 3 pairs of abdominal neuroendocrine cells all co-localize (C, yellow, merge). Scale bar 100 mm. doi:10.1371/journal.pone.0029897.g005 NMU-25 stimulated NMUR 2- and apoaequorin-co-transfected S2 cells (Fig. 3A). NMU-25 has a small but significant effect on [Ca2+]I via capaR (Fig. 3A). We then tested the action of capa-1 at the NMU receptor 2 and show that capa-1 mobilizes [Ca2+]i via NMUR 2 (Fig. 3B). Based on structural and functional similarities, Drosophila hugin has been proposed as an homolog of mammalian NMU [37]. Hugin encodes two peptides: hugin gamma (hugg) and Drm-PK-2, whose C-terminal motifs are related to the insect pyrokinins. Our data show that hugg increases [Ca2+]i via NMUR 2 but to a reduced level compared to capa-1. Interestingly, Drm- PK-1 (capa-3) and -2 do not have effects on either capaR (Fig. 3A) or NMUR 2 (Fig. 3B). However, as Drm-PK-1 and -2 share the - PRLamide signature with hugg (Table 1), we tested their action on CG8795. Stimulation of CG8795- and apoaequorin-co-transfected S2 cells with Drm-PK-1, - 2, and hugg peptides (Fig. 3C), stimulates increased [Ca2+]i levels. However, neither capa-1 nor NMU-25 acts via CG8795. The absence of NMU agonism to CG8795 receptor has been demonstrated [38] and while the homology of NMU to pyrokinins and their respective receptors are higher compared to the capa family of peptides [7,11,35], Drm-PK-2 does not activate the NMUR 2, in contrast to capa-1. Moreover, Fujii et al., have found that Aplysia small cardioactive peptide B (SCPB), which shares the consensus motif, LXXPRX-amide, with neuromedin U, shows a significant agonistic activity to NMR 1 expressed in CHO cells [39]. This indicates that structurally related capa-like peptides, unlike pyrokinins, are able to activate vertebrate NMURs and therefore represent functional NMU homologous. We next investigated a potential role for NMU by challenging, with different concentrations of NMU-25, capaR transfected S2 cells or acutely dissected intact Malpighian tubule. Calcium measure- ments using NMUR 2-transfected S2 cells expressing the apoaequorin transgene (Fig. 3D) showed a robust dose-response curve with an EC50 = 0.91 nM, which is within the nM range obtained with NMUR 2 in HEK293 cells [40]. Furthermore, transgenic tubules in which targeted apoaequorin is expressed in tubule principal cells showed a dose-dependent response to NMU- 25 with an EC50 = 21.8 and 27.1 nM respectively (Fig. 3D) demonstrating that NMU does indeed act on tubules, via endogenous capaR (Fig. 3A). Given that NMU directly activates capaR, we tested the possibility that NMU could have functional roles in vivo. Fluid transport in wild-type tubules is significantly increased by human NMU-25 but not by Drm-PK-1 and -2, or hugg (Fig. 4A). Using transgenic lines for human NMUR 2, in which ectopic expression in tubule was achieved under control of the principal cell-specific c42-GAL4 driver, we show that NMU increases [Ca2+]i in vivo (Fig. 4B). Ectopic expression of human NMUR 2 in tubules using the ubiquitous actin-GAL4 driver also results in significantly elevated fluid transport upon NUM-25 stimulation (Fig. 4C); presumably due to increased [Ca2+]i in principal cells (Fig. 4B). Capa-1 stimulation of NMUR 2 tubules also increases fluid transport rates (Fig. 4D), suggesting that the action of capa-1 at the NMUR 2 receptor (Fig. 3B) can have a physiological role in vivo. Based on the sequence similarities between capa, hugin and NMU peptides, we performed immunocytochemistry using anti- neuromedinU-8 antibody on larval brain. The most strongly immunoreactive cells in the nervous system recognized by the neuromedinU-8 antibody consist of three pairs of ventral neuroendocrine cells in the abdominal neuromeres and a single pair of very large neuroendocrine cells in the subesophagial ganglion (Fig. 5). These immunoreactive cells are identical to the capa neuroendocrine cells [3] demonstrated using anti-capa precursor peptide antibody. It is worth mentioning that anti- NMU-8 also labels hugin neurons (small group of neurons in the SOG) but in a lower extent. Taken together, these data suggest that NMU could be regarded as a functional vertebrate homolog of capa-1. In vertebrates, NMU action modulates several physiological processes feeding and the stress response, as NMU interacts anatomically and functionally with the CRH system which is secreted in response to stressors [41]. There is some evidence that NMU can affect ion transport in the gut [41], but this is the first evidence that NMU can modulate fluid transport by a renal system. Physiological role of capaR in vivo The capaR gene is expressed exclusively in Malpighian tubules in both larvae and adults (Fig. 6A), where capaR expression is up- regulated 42- (adult) and 14.4- fold (larvae) in tubules compared to the whole fly. The putative control region of the capaR gene drives expression of GAL4 in the capaR-GAL4 line: progeny from a cross of capaR-GAL4 and a UAS-GFP line show that fluorescence was specifically detected in the Malpighian tubules, either at the third instar larval or adult stages (Fig. 6B), a result consistent with the microarray data for capaR expression (Fig. 6A). Furthermore, tubule-specific capaR is expressed at the basolateral membrane of the principal and not stellate cells (Fig. 6B–D). Also, typical capa- 1-induced Ca2+ responses occur in principal cells of capaR- PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e29897