G protein-coupled receptors in arthropod vectors: omics and pharmacological approaches to elucidate ligand-receptor interactions and novel organismal functions

Abstract

Regulation of many physiological processes in animals, certainly those controlled by neuropeptide hormones, involves G protein-coupled receptors (GPCRs). Our work focusing on endocrine regulation of diuresis and water balance in mosquitoes and ticks started in 1997 with the kinin receptor, at the dawn of the omics era. After the genomic revolution, we began work on the endocrinology of reproduction in the red imported fire ant. We will use the template of this comparative work to summarize key points about GPCRs and signaling, and emphasize the most recent developments in the pharmacology of arthropod neuropeptide GPCRs. We will discuss omics' contributions to the advancement of this field, and its influence on peptidomimetic design while emphasizing work on blood feeding arthropods.

Figure 1. Omics contribution to arthropod GPCR discovery and functional analyses

Genomes, transcriptomes, peptidomes and cDNA clones (input knowledge) aid in the curation of protein sequences for both GPCRs and peptide ligands. Curated sequences are organized in databases such as the Database for Insect Neuropeptide Research (DINeR [24]). GPCRs are expressed in cell systems for verification of function. If they are highly similar to other deorphanized receptors this constitutes a forward pharmacology approach. Alternatively, they are deorphanized through a reverse-pharmacological approach to discover either endogenous or synthetic ligands or natural small molecule active ligands by high-throughput screening (HTS). Once a ligand is a “hit” on the receptor, as agonist or antagonist, bioassays are performed, or the ligand is used as a tool to probe suspected functions in tissues (in vitro). These processes or activities result in different products, and converge as output knowledge of new ligands, receptor function, comparative endocrinology and receptor-ligand structure models. “Ligand-docking” informs ligand (= lead) optimization, and models are improved after site-directed mutagenesis experiments.

Figure 2. Key elements of GPCR target validation

For chemical validation of a GPCR as a candidate target, the designed or identified ligand that is active on the recombinant receptor is applied in vivo and in vitro in tissues. Bioactivity must be determined, either as mortality or by another adverse biological effect derived from its action as antagonist or superagonist (i.e. paralysis). For genetic validation, “loss of function” experiments most typically, or “gain of function” experiments, must confirm the disruption of receptor function has a measurable effect. Through these processes, we validated stable potent peptidomimetics for, and discovered novel functions of the kinin receptor [73].