Abstract
Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca2+ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.
Highlights
Sulfakinins (SKs) are a family of invertebrate neuropeptides that are involved in the complex regulation of feeding and digestion in insects [1]
Tissues from sexually mature T. castaneum were dissected under a binocular microscope in phosphate buffered saline (PBS) (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.76 mM; pH 7.2) and snap-frozen in liquid nitrogen
A possible disulfide bond can be formed between two cysteine (C125 and C203) residues in extracellular loop (ECL) I and ECL II in the Trica-SKR1
Summary
Sulfakinins (SKs) are a family of invertebrate neuropeptides that are involved in the complex regulation of feeding and digestion in insects [1]. Sulfated peptides that do not have any major modifications in the C-terminal core sequence can activate one or both receptors for at least 50% of the response level reached by sTrica-SK(5–13) at 1 mM. Like drosulfakinin 2, native Trica-SK contains an N-terminal glycine residue, two acidic residues in the N-terminal part of the peptide and the C-terminal nonapeptide FDDYGHMRFa. When compared to the nonsulfated (ns) Trica-SK(5–13), compound 1011[w2a]wp-7-4 activated both Trica-SK receptors to a higher extent.
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