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. prolixus CT/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. melanogaster CT/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 = 200-300nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15nM) 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.
Highlights
Various neurohormone families have been implicated in regulating diuresis in insects
Rhopr-calcitoninlike diuretic hormone (CT/DH)-R1-B and Rhopr-CT/DH-R1-C differ by only 6 nucleotides within their ORF, which results in a 2 amino acid difference between these variants within their intracellular Cterminal domain
We have found that R. prolixus, and perhaps other insects, contain two CT/DH-Rs
Summary
Various neurohormone families have been implicated in regulating diuresis in insects. One such family is the calcitoninlike diuretic hormone (CT/DH) family which is related to the mammalian calcitonin and calcitonin gene-related peptide hormonal system. The first member of this peptide family in insects was isolated and functionally characterized in Diploptera punctata [1]. This peptide was originally termed diuretic hormone 31 (DH31) due to its ability to stimulate Malpighian tubule (MT) secretion in certain insects and due to the fact that it is comprised of 31 amino acids [1,2,3]. In order to elucidate additional physiological roles for these hormones, it is important to identify and characterize their receptors and determine their expression patterns
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