Functional Characterization of Ecdysis Triggering Hormone Receptors (AgETHR-A and AgETHR-B) in the African Malaria Mosquito, Anopheles gambiae

Abstract

Insect ecdysis behavior, shedding off the old cuticle, is under the control of specific neuropeptides with the top command by the ecdysis triggering hormone (ETH). We characterized the ETH receptor (ETHR) of the malaria mosquito, Anopheles gambiae, by manual annotation of the NCBI gene (AGAP002881) and functional analysis, using a heterologous expression system in a mammalian cell line. The two splicing variants of ETHRs, ecdysis triggering hormone receptors (AgETHR-A and AgETHR-B), a conserved feature among insects, included of four (552 aa) and five exons (635 aa), respectively. The main feature of manual annotation of the receptor was a correction of N-terminal and exon-intron boundaries of an annotated gene (AGAP002881). Interestingly, the functional expression of the receptor in Chinese hamster ovary cells required modification of the transcription initiation site for mammalian Kozak consensus. In the calcium mobilization assay using the heterologous expression of each receptor, AgETHR-B showed a higher sensitivity to AgETH-1 (28 times) and AgETH-2 (320 times) than AgETHR-A. The AgETHRs showed specificity only to the ETH group of peptides but not to other groups carrying the C-termini motifs as PRXamide, such as pyrokinin1/DH and pyrokinin2/PBAN. Ecdysis triggering hormone receptors (AgETHR-B) responded to different ETH variants of other insect species more promiscuously than AgETHR-A.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation

“…We tested AgETH1 on the receptor AgETHR‐A because they are the pair showing relatively high specificity to only mosquito ETHs based on our earlier study (Jindal et al ., 2021 ). The specificity of AgETHR‐A was important to discriminate the essential amino acid residues involved in the receptor activation.…”

“…ETH sequence alignments were using the sequences for 81 insect ETH captured in the DINeR (Yeoh et al ., 2017 ). The procedure for gene cloning and heterologous expression of AgETHR‐A1 was previously described (Jindal et al ., 2021 ). We chose AgETHR‐A, not the isoform AgETHR‐B, for the study because the former provided higher specificities to the AgETH‐1 in the preliminary study (Jindal et al ., 2021 ).…”

“…The procedure for gene cloning and heterologous expression of AgETHR‐A1 was previously described (Jindal et al ., 2021 ). We chose AgETHR‐A, not the isoform AgETHR‐B, for the study because the former provided higher specificities to the AgETH‐1 in the preliminary study (Jindal et al ., 2021 ). Briefly, the receptor clone was expressed in Chinese Hamster Ovarian cell (CHO‐K1) that contains apoaequorin and Galpha 16.…”

“…1 ). In this study, we modeled the ETH1 of African malaria mosquito Anopheles gambiae (AgETH1, 17mer SESPGFFIKLSKSVPRI‐NH 2 ) (Park et al ., 2002b ; Park et al ., 2003 ; Jindal et al ., 2021 ). We investigated the structural components of the ETH for activation of the AgETHR‐A.…”

Characterization of Capa Peptide and Its Receptor in Bemisia Tabaci

“…The GPCRs have been reported for ecdysis 11,12 , reproduction 13,14 , diapause 15,16 , feeding 17 , pheromone biosynthesis 18,19 , expressing detoxi cation enzyme 20 , diuresis 21,22,23 , anti-diuresis 24,25 , etc. One of the most vital physiological processes for sap-feeding insects like aphids and bugs is diuresis, which helps it in maintaining the hemolymph ion-water balance and excreting out the metabolic waste (sodium, chloride, potassium, toxins, water) through absorption by Malpighian tubules which would otherwise prove lethal 24 .…”