Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans

Insight into mosquito GnRH-related neuropeptide receptor specificity revealed through analysis of naturally occurring and synthetic analogs of this neuropeptide family

“…Similar observations have been made with AKH analogs tested using both in vivo and in vitro assays whereby complete removal of the N-terminal pyroglutamate abolishes activity of the AKH analog whereas the alternatively blocked N-terminus (i.e. N-Acetyl-Ala), or even simply glutamate or glutamine that can spontantenously undergo cyclization (Schilling et al, 2008), were shown to have improved activity, albeit lower than the native AKH (Caers et al, 2016; Gäde and Hayes, 1995; Marco and Gäde, 2019b). Lastly, although alanine substitution for valine in the second position led to a relatively minor effect on ACPR activation (~70-fold reduced activity), the internally truncated analog omitting valine within the N-terminal region elicited no activation of the ACPR, indicating the spacing between the more critical residues, namely pyroglutamate and polar threonine, is essential for ACP peptide activity on the ACPR.…”

“…Previous studies examining free acid analogs of AKH (including both in vitro and in vivo assays) have demonstrated that in some cases the absence of a blocked C-terminus is less critical (Caers et al, 2012; Caers et al, 2016; Lee et al, 1996), whereas other investigations have revealed the presence of an amidated C-terminus to be very important with poor or no responses with analogs lacking this feature (Gäde and Hayes, 1995; Gäde, 1990). These observed differences in response to key structural elements, including the normally amidated C-terminus of AKH, may reflect the occurrence of inter- and intra-species receptor subtypes which may be differentially sensitive to these modifications (Caers et al, 2016; Gäde and Hayes, 1995), and could also point to the well documented phenomenon that some species have AKH receptors that are more promiscuous whereas others are very strict in the structural characteristics of their particular ligand (Caers et al, 2012; Gäde, 1990; Marchal et al, 2018). This latter scenario is more in-line with observations for the A. aegypti ACP system where only a single functional receptor variant occurs (Wahedi and Paluzzi, 2018), which we show herein does not tolerate the absence of the C-terminal amidation and is thus of high importance for the ACP system.…”

“…Interestingly, structure-activity studies on the two tsetse fly ( G. morsitans ) AKH receptors using synthetic analogs of the pair of endogenous AKHs identified the most critical sites being the aromatic residues in positions four and eight, but the next most important features included positions 1-3 from the N-terminus with activity of these analogs eliciting only 20-50% activity compared to the two endogenous AKH peptides (Caers et al, 2016). On the other hand, the region of the G. morsitans AKH diplaying increased permissiveness for substitutions included positions six and seven, where glycine substitutions of these sites resulted in these analogs having between 65-97% activity compared to the efficacy of the native AKHs.…”

Characterization of the adipokinetic hormone receptor of the anautogenous flesh fly, Sarcophaga crassipalpis

“…They still allow this peptide to bind and activate the receptor although much higher concentrations are needed. Substitutions by amino acids with different nature are described to almost completely abolish interaction with AKH receptors (Caers et al, 2012(Caers et al, , 2016.…”

“…The aequorin-bioluminescence assay was used to measure the calcium release in transfected CHO WTA11 cells upon ligand exposure (as also described in Caers et al, 2016;Verlinden et al, 2015). Transfected cells were detached using 0.2% EDTA-PBS solution and collected in 10 ml DMEM/F12.…”