Mechanism and Function of Drosophila capa GPCR: A Desiccation Stress-Responsive Receptor with Functional Homology to Human NeuromedinU Receptor

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.

Insect capa neuropeptides impact desiccation and cold tolerance

“…Both these phenotypes were capa-dependent and could be reversed with active capa peptide analogs. CapaR is localized exclusively in Malpighian tubule principal cells, and we have shown previously that targeted knockdown of capaR mediates whole-fly desiccation tolerance (25). We also show that knockdown of capaR significantly increases CCR (Fig.…”

“…In dipteran insects, capa is diuretic, acting on the Malpighian tubules to modulate cell-signaling and ion-transport pathways (24). CapaR, the G protein-coupled receptor for the capa peptides, is localized exclusively in tubule principal cells, and we have shown previously that targeted knockdown of capaR increases whole-fly survival under desiccation stress caused by reduced capa-stimulated diuresis (25). Capa/capaR signaling is functionally conserved in the tubules of dipteran disease vector species including mosquitoes and tsetse flies (24) and is of increasing interest as a target for insect control through the design of peptide mimetic analogs (26).…”

“…Thus, the reduced rate of water loss in capaknockdown flies may be caused by combined reduction in respiratory, cuticular, and excretory water loss. However, the direct action of capa peptides on capaR exclusively expressed in the Malpighian tubules (25) suggests that excretory water loss may contribute more to desiccation tolerance than previously suspected.…”

“…2A) (22). The neurite network of the two anterior Va neurons pairs is confined to the dorsal ganglion and forms a neurohemal area, suggesting release of capa peptides into the hemolymph, whereupon they activate capaR, the G protein-coupled receptor for Drm-capa-1 and Drmcapa-2 (31), which is localized exclusively in Malpighian tubule principal cells (25).…”

“…Recent work indicates that CCR is influenced primarily by the magnitude of ion disruption in the gut during cold exposure and the time taken to restore ion and water balance to regain motor control, potentially via ion-motive epithelial ATPases (12). Vacuolar H + -ATPase (V-ATPase) is most highly expressed in Malpighian tubule principal cells (37), which also uniquely express capaR (25). Capa increases V-ATPase activity by elevating mitochondrial calcium and ATP levels (38), increasing tubule diuresis.…”