Mechanism and Function of Drosophila capa GPCR: A Desiccation Stress-Responsive Receptor with Functional Homology to Human NeuromedinU Receptor Selim Terhzaz1*, Pablo Cabrero1, Joris H. Robben2, Jonathan C. Radford1, Brian D. Hudson 1 , Graeme Milligan1, Julian A. T. Dow1, Shireen-A. Davies1* 1 Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom, 2 Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Abstract The capa peptide receptor, capaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-capa-1 and -2 (capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptide family are of interest. Capa peptide induces calcium signaling via capaR with EC50 values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. capaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal - FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm- PK-2 and hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca2+]i) levels via capaR. In vivo, NMU-25 increases [Ca2+]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca2+]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca2+]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, capaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly. Citation: Terhzaz S, Cabrero P, Robben JH, Radford JC, Hudson BD, et al. (2012) Mechanism and Function of Drosophila capa GPCR: A Desiccation Stress- Responsive Receptor with Functional Homology to Human NeuromedinU Receptor. PLoS ONE 7(1): e29897. doi:10.1371/journal.pone.0029897 Editor: Leo T. O. Lee, University of Hong Kong, Hong Kong Received July 20, 2011; Accepted December 8, 2011; Published January 11, 2012 Copyright: 2012 Terhzaz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) project grant BB/G020620 to SD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Shireen.Davies@glasgow.ac.uk (S-AD); Selim.Terhzaz@glasgow.ac.uk (ST) Introduction Drosophila is an excellent model for insect pest species especially the flies [1,2]. As insects can withstand desiccation so well, in general, the detailed understanding of mechanisms of desiccation or water stress in vivo is a potential route for intervention. Insect neuropeptides, including diuretic peptides and their cognate receptors, are a key research area for potential novel routes for such control. D. melanogaster capa- 1 and -2 (Drm-capa-1 and -2) neuropep- tides act on the Malpighian tubules to increase fluid transport [3]. Tubules are transporting epithelia equivalent to vertebrate kidney and liver, and regulate water and ion homeostasis, and detoxification [4]. Capa-1 and capa-2 (and the related Manduca sexta CAP2b) are the only known nitridergic peptides in insects, acting via elevation of intracellular calcium, ([Ca2+]i) and activation of nitric oxide/cGMP signaling in tubule principal cells [3]. Capa peptides show a complex mode of action - in addition to stimulation of NO/cGMP signaling, capa-1 also modulates calcium signaling in the mitochondria [5], Golgi and peroxisomes [6]. There is also close conservation between capa peptide structure [7,8] and capa-induced signaling cascades in tubules of the disease vectors Anopheles, Aedes and Glossina (tsetse fly) [9,10]. Here, we demonstrate precise kinetics for capa-induced [Ca2+]I signaling; and desensitization and internalization of capaR via b- arrestin. The capa receptor (capaR) [11,12] is a G-protein coupled receptor (GPCR) and member of the PRXamide peptide receptor family. The PRXamide C-terminal motif occurs in several invertebrate and vertebrate peptides [12]. There is significant interest in identification of vertebrate homologs of insect neuropeptides, as increasingly, key novel physiological functions eg., in regulation of feeding behaviour, have been ascribed to insect neuropeptides [13]. Thus, potential homologous neuropeptide agonists of capaR are of significant interest. We identify human neuromedin U as a putative functional homolog of capa-1, via cell- based assays, in vivo assays using transgenic human NMU receptor 2 flies, and immunocytochemical studies in the larval nervous system. PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29897