Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Yang, Yunlong; Bajracharya, Prati; Castillo, Paula; Nachman, Ronald J.; Pietrantonio, Patricia V.

doi:10.1016/j.ygcen.2013.09.001

Cited by 20 publications

(26 citation statements)

References 55 publications

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“…Negative controls, mock transfected ( Figure 5D–F ) and the stable cell line Si sNPFR-C6E8 incubated with pre-immune rabbit serum ( Figure 5J–L ) did not show any signal, as expected. The Rhimi -CAP2b-R #19 cell line which stably expresses a HA-tagged tick CAP 2b receptor [55] , was used as positive control ( Figure 5G–I ). The Si sNPFR-C6E8 cell line was also stained with an anti-α tubulin antibody as an additional positive control; this showed the distinctive filamentous signal in the cell cytoplasm.…”

Section: Resultsmentioning

confidence: 99%

“…For expression of this receptor cDNA in CHO-K1 cells, the clone designated p Si sNPFR#16 (in the TOPO vector pCR2.1, Invitrogen) containing the full-length cDNA of the Si sNPFR was used as the template for a PCR to add a nine amino acid residue haemaglutinin tag (HA-tag; YPYDVPDYA) at the receptor N-terminus, following published procedures [53] – [55] . For this, three overlapping forward primers were designed ( Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…For immunocytochemistry experiments, 1×10 5 cells were sown over sterile circular coverslips (Fisher Scientific, Pittsburgh, PA, USA), that were placed in, 12-well plates. Cells, mock-transfected with vector pcDNA3.1(-) only, and Si sNPFR-C6E8, were allowed to attach onto the coverslip after incubation, for 24 h. Cells were then processed for immunolocalization of the HA-tag-labeled receptor according to methods in Yang et al [55] . Briefly, cells were prefixed in freshly prepared 4% paraformaldehyde (PFA) in F12 K culture medium with 10% fetal bovine serum and Geneticin.…”

Section: Methodsmentioning

confidence: 99%

“…Si sNPFR-C6E8 cells were incubated with preimmune rabbit serum (1∶2000) as negative controls, and also incubated with rabbit anti α-tubulin IgG (1∶50 dilution; Abcam, Cambridge, MA, USA) as a positive control for immunostaining. As negative and positive controls for the HA-tag antibody staining, mock-transfected cells and RmCAP2bCCL#19 [55] cell line expressing an HA-tag neuropeptide receptor were incubated with the anti HA-tag primary antibody, respectively.…”

Section: Methodsmentioning

confidence: 99%

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The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

Bajracharya

Pietrantonio

2014

PLoS ONE

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Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

Bajracharya

Pietrantonio

2014

PLoS ONE

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“…The current study provides definitive evidence supporting the importance of this anti-diuretic hormone system in the disease vector mosquito, A. aegypti , by characterization and functional deorphanization of an anti-diuretic hormone receptor that is highly enriched in the MTs and demonstrates high selectivity for the mosquito CAPA neuropeptides. Previous studies have functionally deorphanized a number of CAPA receptor orthologs in other insects including dipterans 44,57,82,83 , lepidopterans 84 , coleopterans 85 , hemipterans 86 , as well as in the southern cattle tick 87 . Here, we have functionally validated the specific ligands of the elusive A. aegypti CAPA receptor demonstrating that two of the peptides encoded by the mosquito CAPA gene 50 , Aedae -CAPA1 and –CAPA2, potently activate this receptor leading to calcium signalling that elicits a bioluminescent response.…”

Section: Discussionmentioning

confidence: 99%

An anti-diuretic hormone receptor in the human disease vector,Aedes aegypti: identification, expression analysis and functional deorphanization

Sajadi

Uyuklu

Lajevardi

et al. 2019

Preprint

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Insect CAPA neuropeptides, which are homologs of mammalian neuromedin U, have been described in various insect species and are known to influence ion and water balance by regulating the activity of the Malpighian ‘renal’ tubules (MTs). A number of diuretic hormones have been shown to increase primary fluid and ion secretion by the insect MTs and, in the adult female mosquito, a calcitonin-related peptide (DH31) also known as mosquito natriuretic peptide, increases sodium secretion at the expense of potassium to remove the excess salt load acquired upon blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having inhibitory activity against select diuretic factors and being particularly potent against DH31-stimulated diuresis. In the present study, we have functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr). Expression analysis by quantitative PCR indicates the receptor is highly enriched in the MTs, and fluorescent in situ hybridization confirms expression within principal cells. Characterization using a heterologous system demonstrated the receptor was highly sensitive to mosquito CAPA peptides. In adult females, AedaeADHr transcript knockdown using RNAi led to the abolishment of CAPA-peptide induced anti-diuretic control of DH31-stimulated MTs. The neuropeptidergic ligand is produced within a pair of neurosecretory cells in each of the six abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs (known as the perivisceral organs), where these neurohormones are released into the open circulatory system of the insect. Furthermore, pharmacological inhibition of PKG/NOS signalling abolished the anti-diuretic activity of AedaeCAPA-1, which collectively confirms the role of cGMP/PKG/NOS in this anti-diuretic signalling pathway.SignificanceInsects are by far the most successful and abundant group of organisms on earth. As a result of their small size, insects have a relatively large surface area to volume ratio, raising the potential for rapid gain or loss of water, ions and other molecules including toxins – a phenomenon that applies to insects living in both aquatic and terrestrial environments. In common with many other organisms, hormones are key regulators of the excretory system in insects, and numerous factors control the clearance of excess water and ions (i.e. diuretics) or retention of these elements (i.e. anti-diuretics). Here we characterized an endogenous anti-diuretic hormone receptor in the human disease vector, Aedes aegypti, demonstrating its expression is highly enriched in the Malpighian ‘renal’ tubules and is necessary for eliciting anti-diuretic control of this key component of the mosquito excretory system.

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The PRXamide Neuropeptide Signalling System

Jurenka

2015

Advances in Insect Physiology

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Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Cited by 20 publications

References 55 publications

The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

An anti-diuretic hormone receptor in the human disease vector,Aedes aegypti: identification, expression analysis and functional deorphanization

The PRXamide Neuropeptide Signalling System

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