Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors (original) (raw)

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the threedimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety. Insect sulfakinin (SK) signaling is active in a variety of biological processes in insects, such as the regulation of feeding 1-5 and muscle contraction 6-8. SK signaling consists of SK peptides, SK receptors (SKRs) and other molecules. The interaction of SK peptides and SKRs initiates the SK signaling transduction. SK peptides share a conserved carboxyl-terminal amino acid sequence YGHMRF-NH 2 with different amino-terminal extensions 9. Two forms of SK peptides are often found in insects as sulfated SK (sSK) and nonsulfated SK (nsSK), depending on the presence of a sulfate group on the tyrosyl residue 8. SKRs are G-protein coupled receptors (GPCRs) that can convert the extracellular signals into intracellular signals 10. Insect SK signaling is found to be homologous to the cholecystokinin (CCK) signaling in humans since they show homology over their components, signaling transduction and functions 2,6,7,9,11,12. Human CCK signaling has been investigated extensively. It involves the CCK peptides and CCK receptors 13. CCK peptides exist in two forms: sulfated CCK (sCCK) and nonsulfated CCK (nsCCK). In human CCK signaling, two CCK receptors (CCKRs), namely CCK1R and CCK2R, show different affinities to sCCK and nsCCK. CCK1R is activated by sCCK 500-to 1000-fold more than by nsCCK, while CCK2R responds to both sCCK and nsCCK similarly 13. The ligand binding sites in CCKRs have been examined via site-directed mutagenesis 14-18 , photoaffinity labeling 19,20 and molecular modeling 16-19. Several amino