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First published online March 21, 2005
Journal of Experimental Biology 208, 1239-1246 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01529

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A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling

Erik C. Johnson^1,*, Orie T. Shafer¹, Jennifer S. Trigg¹, Jae Park², David A. Schooley³, Julian A. Dow⁴ and Paul H. Taghert^1,

¹ Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA
² Department of Biochemistry, Cell and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA
³ Department of Biochemistry, University of Nevada, Reno, NV 89557, USA
⁴ Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK

View larger version (9K): [in a new window]   Fig. 1. The CG17415 receptor responds to DH31 application but requires the presence of the RCP. Cells were transfected with CG17415 and CRE-luc DNA and assayed for increases in luciferase activity as a result of peptide exposure. Values are means ± S.E.M. from six wells derived from two independent transfections, and expressed as a percentage of vehicle-treated cells. (A) In NIH 3T3 cells, 1 µmol l–1 DH31 elicited a significant difference in cAMP levels (P<0.007, two-tailed t-test; Excel) compared to vehicle controls, and did not respond to any of 29 other native and heterologous peptides. (B) Dose–response curve of DH31 sensitivity in HEK cells expressing CG17415 and CG4875 (dRCP) DNA. The maximum response was twofold and the estimated EC50 is 0.5 µmol l–1. (C) Dose–response curve of DH31 sensitivity in HEK cells expressing CG17415 and human RCP. Note the much larger (10-fold) difference in signal intensity, and an estimated EC50 of 116 nmol l–1.

View larger version (17K): [in a new window]   Fig. 2. RAMP subtype expression does not alter the DH31-R pharmacological profile. HEK-293 cells were transfected with CG17415, CRE-luc and either no RAMP DNA (hatched), RAMP1 (filled) or RAMP2 (unfilled) (at a ratio of 5:1:1 CG17415: RAMP:CRE-luc), but without RCP. Value are means ± S.E.M. from six wells (drawn from two replicate experiments) and expressed as a percentage of vehicle-treated cells. Peptides were all tested at 1 µmol l–1 concentrations and only DH31-treated cells showed significant increases in luciferase activity levels as compared to controls. Dromyosuppressin (DMS), adipokinetic hormone (AKH), crustacean cardioactive peptide (CCAP), ecdysis triggering hormone (ETH), and pigment dispersing factor (PDF) were purchased from Multiple Peptide Systems (San Diego, CA, USA). Allatostatin A (AstA), allatostatin C (AstC) and DPKQDFMRFamide (FMRF) were purchased from BACHEM (King of Prussia, PA, USA). Proctolin (PROC) and corazonin (CRZ) were purchased from Sigma (Saint Louis, MO, USA). Diuretic hormone 31 (DH31) was from batch the synthesis of which has been reported previously (Coast et al., 2001). Diuretic hormone 44 (DH44) was synthesized by Syngenta Biotechnology (Research Triangle Park, NC, USA) and leucokinin (LK) were purchased from Invitrogen. Allatostatin B (AstB) was provided by Jan Veenstra, NPF by Joe Crim, Accessory Peptide 99B (SP) by Erik Kubli, ITP, PK2, and hugin peptides by Michael Adams, IPNa and MTYa by Liliane Schoofs, and amnesiac (AMN) peptide from Scott Waddell.

View larger version (102K): [in a new window]   Fig. 3. Desensitization of Drosophila DH31-R1 requires accessory proteins. (A) Two typical HEK cells following transfection with ß-Arr2-GFP, RAMP1, RCP and CG17415, prior to peptide application. The cells display a uniform cytoplasmic distribution of fluorescence. (B) A different cell 20 min following application of 1 µmol l–1 DH31. Note prominent rearrangement of fluorescence in large vesicles throughout the cell. Cells were transfected with DNA at a ratio of 5 (CG17415): 1 (RAMP1): 1 (RCP): 1 (ßarr2-GFP).

View larger version (31K): [in a new window]   Fig. 4. DH31-R expression in the principal cells of Malpighian tubules. Malpighian tubules from individual progeny of a c724::GAL4 cross to UAS::lacZ were double stained with antibodies for CG17415 and ß-galactosidase (center). Note the specific labeling of the stellate cells (left, arrow) with the anti-ß-gal antibody and the complementary labeling in the principal cells (right, arrow) of the tubules with anti-CG17415.

View larger version (94K): [in a new window]   Fig. 5. Expression of the DH31-R1 receptor in the larval brain reveals a convergence with DH44-R1 and labels corazonin neurons. (A–C) Three different Z-series through the CNS reveals expression of (A) DH31-R1 (CG17415), (B) DH44-R1 (CG8422) and (C) corazonin in the brain and ventral nerve cord. (Left) Expression of DH31-R1 (CG17415) in the CRZ-expressing neurons of the larval brain (top and asterisk) and in the ventral nerve cord (bottom and arrow). (Right) Expression of DH44-R1 (CG8422) in the CRZ-expressing neurons of the larval brain (top and asterisk) and in the ventral nerve cord (bottom and arrow).

View larger version (109K): [in a new window]   Fig. 6. Adult co-expression of two different DH receptors in neurons producing the neuropeptide corazonin. (A) The central brain of adult flies expressing lacZ under the control of CRZ::GAL4 (left), and double-labeled for anti-ß-gal and for anti-CG17415 (DH-31-R1). All CRZ neurons express CG17415 (arrowhead) but there were two to four CG17415 neurons in the dorsal brain that did not express CRZ. (B) Brains from the same cross, double-labeled for anti-ß-gal and anti-CG8422 (DH-44-R1). As for CG17415, all CRZ neurons express CG8422 (arrowhead). In addition, three to four CRZ-negative, 8422-positive neurons were present. (C) Diagram of the adult brain CNS summarizing the large-scale coincidence of CG17415, CG8422 and CRZ expression patterns.

View larger version (33K): [in a new window]   Fig. 7. A model of convergent neuropeptide DH31 (CLR) and DH44 (CRF-R) signaling pathways. DH31-R (red) and DH44-R (blue) signaling pathways are co-expressed by principal cells of the tubule (top), and onto CRZ-containing neurons of the CNS (bottom). Both pathways elicit cAMP increases; the DH31-R1 utilizes a RCP accessory protein. We show DH31 and DH44 neurons as distinct cell populations in this model (see Fig. S2 in supplementary material), but these may be partially overlapping. M. tubule, Malphigian tubule.