Molecular characterization and role of ecdysone membrane receptor

我们阐明了对20E的早期反应基因在五龄至肠道清洗后两天细胞死亡处死前的丝腺中的基因表达的发育特征。此外，我们还使用肠道净化幼虫的前丝腺在体外显示了20E的基因响应。体内和体外结果表明，异源二聚体ECR-B1和USP-2可能是导致细胞死亡的原因，同时也表明E74、E75、BHR3和BR-C亚型参与了细胞死亡，而FTZ-F1没有参与。膜蜕皮激素受体的基因克隆尚未成功。我们从药理学上研究了从mEcR到细胞反应的信号通路，即细胞凝聚、核凝聚、DNA片段化和核片段化。第二信使可能是G-蛋白偶联受体，与G-αQ相关，继而激活磷脂酶c-β，产生3-磷酸肌醇(IP_3)，并可能通过IP_3受体从内质网上释放Ca~(2+)，激活蛋白激酶C(PKC)和类半胱氨酸蛋白酶。这一信号通路最终导致核碎裂和核碎裂。核缩合受钙调蛋白和钙调蛋白依赖蛋白激酶II(CaMK-II)的不同途径调节。然而，该途径不被Ca&lt；2+&gt；激活，因此G-αQ是否参与该途径尚不清楚。此外，钙调素和CaMK-II的抑制剂影响了细胞核和DNA片段化的发生，表明Caspase3样蛋白水解酶的激活不是简单地依赖于GPCr/PLC-β/IP3/Ca&lt；2+&gt；/PKC信号通路。

We clarified developmental profiles of gene expression of early response genes to 20E in the anterior silk glands during the fifth instar up to the time of cell death execution two days after gut purge. Also, we showed the gene response to 20E in vitro using anterior silk glands of gut-purged larvae. The in vivo and in vitro results indicated that a heterodimeric EcR-B1 and USP-2 may be responsible for the cell death Results also indicated involvement of E74, E75, BHR3 and BR-C isoforms, but not Ftz-F1. We are not succeeded in gene cloning of the putative membrane ecdysone receptor yet. We examined pharmacologically the signaling pathway from mEcR to cellular responses, i.e. cell condensation, nuclear condensation, DNA fragmentation and nuclear fragmentation. Ca<2+> acts as the second messenger The mEcR is suggested to be a G-protein coupled receptor (GPCR) associated with Gαq, followed by a serial activation of phospholipase c-β, generation of inositol 3-phosphate (IP_3), and release of Ca<2+> from endoplasmic reticulum probably through IP3 receptor Then, Ca<2+> activates protein kinase C (PKC) and caspase 3-like protease. This signaling pathway culminates in nuclear fragmentation and nuclear fragmentation. Nuclear condensation is regulated by a different pathway involving calmodulin and calmodulin-dependent protein kinase II (CaMK-II). However, this pathway was not activated by Ca<2+>, and therefore it is unknown whether Gαq is involved in this pathway. In addition, inhibitors of calmodulin and CaMK-II affected the occurrence of nuclear and DNA fragmentations, indicating the caspase 3-like protease activation does not depend simply on the signaling pathway of GPCR/PLC-β/IP3/Ca<2+>/PKC.