Insights into adipokinetic hormone/corazonin-related peptide receptor specificity and key residues for its activation in the human disease vector Aedes aegypti mosquito

Adipokinetic hormone/corazonin-related peptide (ACP) and adipokinetic hormone (AKH) are two neuropeptides that demonstrate homology to the vertebrate gonadotropin-releasing hormone (GnRH). Despite the structural similarity and the close evolutionary relationship between the ACP and the AKH, their signaling systems function independently. To date, the role of ACP and its receptor (ACPR) remains unclear in the Aedes aegypti mosquito. Structure-activity relationships (SARs) are often carried out on peptide ligands to determine critical residues for bioactivity and receptor activation; however, residues and features necessary for ligand binding and specificity in the receptors themselves are less studied. Herein, this study focuses on the ACP and AKH signaling systems and examines structural features of their receptors critical for conferring activation and ligand specificity. Firstly, to determine the specific ACPR regions most critical for ligand fidelity and specificity, ACPR chimeras were created by singly replacing each of the three ACPR extracellular loops (ECLs) in their entirety and incorporating the corresponding ECLs from the AKH receptor (AKHR). Heterologous functional assays determined that the three ACPR ECL chimera receptors with complete replacement of full individual ECLs showed no response to either ACP or AKH. These results suggest that the complete replacement of each individual extracellular loop is detrimental to ligand binding and recognition. Secondly, through a more targeted approach, we aimed to determine specific residues critical for functional ligand-binding by substituting only select highly conserved residues within the three ECLs of the ACPR with those from the AKHR. Modifications of specific and highly conserved residues in these ACPR ECLs chimeras suggest that the third extracellular loop contains the most critical residues necessary for ACP binding and receptor activation. In addition, the combination of two selectively-modified ACPR ECLs demonstrated a significant decrease in response to ACP compared to the native ACPR response. Interestingly, combining all of the ACPR ECLs chimeras together resulted in a significant decrease in response to ACP compared to native ACPR. Relatedly, a significantly increased response to AKH was observed in the receptor chimera combining selected modifications in all three ECLs compared to native ACPR. Hence, the particular residues essential for ACP ligand interaction were identified due to the detrimental effect that occurred in ACPR activation after the selective modification of crucial residues localized within the three extracellular domains of the receptor. These data provide key insight into how these two closely related neuropeptidergic systems maintain functional independence in the mosquito A. aegypti as well as other insects.