The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

Abstract

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.

Inhibitory Role of the Mud Crab Short Neuropeptide F in Vitellogenesis and Oocyte Maturation via Autocrine/Paracrine Signaling

“…To date, several insect sNPF receptors have been cloned and characterized ( 33 , 41 , 59 – 68 ). Among these receptors, the sNPFRs from D. melanogaster ( 67 , 68 ), A. gambiae ( 60 ), B. mori ( 41 , 61 ), S. gregaria ( 33 ), A. aegypti ( 62 ), S. invicta ( 63 ), G. m. morsitans ( 64 ), and the oriental fruit fly, Bactrocera dorsalis (Hendel) ( 65 ) have been deorphanized by ligand-receptor activation of signaling in a heterologous cell expression system, with EC 50 values in the nanomolar range. Compared to the high sensitivity of insect sNPFRs, Sp -sNPFR in our study had a relatively higher half-maximal effective concentration (EC 50 ) in the HEK293T expression system.…”

Brain gene expression analyses in virgin and mated queens of fire ants reveal mating‐independent and socially regulated changes

“…Additionally, some of our pioneering work focused on a G‐protein‐coupled receptor (GPCR) signaling system, the sNPF (sNPY) system. We showed that mated queen starvation resulted in the significant reduction in sNPF receptor transcripts in queen brains suggesting this system links nutrition and reproduction (Ament, Velarde, Kolodkin, Moyse, & Robinson, ; Bajracharya, Lu, & Pietrantonio, ; Chen & Pietrantonio, ). Importantly, the sNPF receptor protein is present in the brain of both virgin and mated queens but it is only expressed in oocytes in ovaries of mated queens (Lu & Pietrantonio, ).…”

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

“…1, center, Processes). Functional analyses of arthropod receptors utilizes calcium mobilization assays (using bioluminescence [27] or fluorescence), or by measurements of cAMP [28]. These are G-protein dependent assays often used in academic settings for forward pharmacology and can be low-throughput or high-throughput (Fig.…”

“…It was not until the release of the fire ant genome and confirmation by cloning a cDNA encoding an unusual sNPYa that we succeeded: Two potential ligands predicted from the translated sequence activated the recombinant receptor by decreasing cAMP in CHO-K1 cells. Mass spectrometry of queen brain confirmed the presence of one peptide fragment [28].…”