title: Isolation and functional characterization of calcitonin-like diuretic hormone receptors in Rhodnius prolixus.
abstract: Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixusCT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogasterCT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC50 = 200-300 nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15 nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.
authors: Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada ; Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors: Meet
authors: M
authors: Zandawala
authors: Shizhong
authors: S
authors: Li
authors: Frank
authors: F
authors: Hauser
authors: Cornelis J P
authors: CJ
authors: Grimmelikhuijzen
authors: Ian
authors: I
authors: Orchard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004231:Diuresis / Q000502:physiology*; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012239:Rhodnius
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007301:Insect Hormones; D012333:RNA, Messenger; D018003:Receptors, Calcitonin