Study On Signal Transduction Mechanism Of Premenstrual Thymosin Receptor In Silkworm

In insects, molting and metamorposis are strictly regulated by molting hormones, known as ecdysteroids. Ecdysteriod synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide BmPTSP, isolated from the larval brain of Bombyx mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide has been identified as a receptor for BmPTSP. However, details on the signaling pathways and physiological functions of this receptor have remains elusive. Here, we report the functional characterization of the BmPTSPR/SPR using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin or AKH-induced CRE-driven luciferase activity in a manner comparable to synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca2+ mobilization and internalization than did BmPTSPl. Additionally, FAM fluorophore-labeled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabeled DmSP, but not by unlabeled BmPTSP1 and, in vice, the binding of FAM-BmPTSP3 was blocked by unlabeled BmPTSP3, but not by unlabeled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx nonvisual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSPl is likely an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gi/o-dependent cAMP production and away from Ca2+ mobilization and arrestin recruitment.