Abstract
The recently discovered adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic (AKH) hormones, which all demonstrate homology to the vertebrate gonadotropin-releasing hormone (GnRH). To date, the function of the ACP signaling system remains unclear. In the present study, we molecularly identified the complete open reading frame encoding the Aedes aegypti ACP receptor (ACPR), which spans nine exons and undergoes alternative splicing giving rise to three transcript variants. Only a single variant, AedaeACPR-I, yielding a deduced 577 residue protein, contains all seven transmembrane domains characteristic of rhodopsin-like G protein-coupled receptors. Functional deorphanization of AedaeACPR-I using a heterologous cell culture-based system revealed highly-selective and dose-dependent receptor activation by AedaeACP (EC50 = 10.25 nM). Analysis of the AedaeACPR-I and AedaeACP transcript levels in all post-embryonic developmental stages using quantitative RT-PCR identified enrichment of both transcripts after adult eclosion. Tissue-specific expression profiling in adult mosquitoes reveals expression of the AedaeACPR-I receptor transcript in the central nervous system, including significant enrichment within the abdominal ganglia. Further, the AedaeACP transcript is prominently detected within the brain and thoracic ganglia. Collectively, these results indicate a neuromodulator or neurotransmitter role for ACP and suggest this neuropeptide may function in regulation of post-ecdysis activities.
Highlights
Neuropeptides are structurally and functionally the most diverse class of signaling molecules that function in intercellular communication in multicellular organisms[1]
Characterization of the adipokinetic hormone/corazonin-related peptide (ACP) signaling system in R. prolixus, Tribolium castaneum, and A. gambiae revealed that the adipokinetic hormone (AKH), CRZ, and ACP receptors were only activated by their corresponding ligand, and are suggested to be independent signaling systems[17,25]
In both R. prolixus and A. gambiae, it was shown that CRZ cannot activate either ACPR or AKH receptors (AKHR) and neither AKH nor ACP can activate CRZR, which indicates that the AKH/AKHR and ACP/ACPR signaling systems are more closely related to one another[17,25]
Summary
Gene specific forward and reverse primers were designed using Primer 3 in Geneious Software (Biomatters Ltd, Auckland, New Zealand) based on a predicted incomplete A. aegypti ACPR sequence (XM_001653870) described previously[17] to amplify a 975 bp partial fragment using Q5 High Fidelity DNA Polymerase (New England Biolabs, Whitby, On) and whole adult female A. aegypti cDNA as template. After successful validation of the cloned partial sequence, primers were designed (as mentioned above) to perform 5′ and 3′ rapid amplification of cDNA ends (RACE)-PCR utilizing the Clontech SMARTer 5′/3′ RACE Kit (Takara BIO USA Inc, CA, USA). Initial attempts at 5′RACE using the 5′CDS primer (provided in the RACE kit) for first-strand cDNA synthesis and subsequent PCR with the SeqAmp DNA Polymerase (Takara BIO USA Inc, CA, USA) was not successful for this target.
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