Molecular characterization and role of ecdysone membrane receptor

我们阐明了前丝腺中20E早期应答基因在5龄至肠道净化后2天细胞死亡执行时的基因表达发育谱。此外，我们还利用肠道净化幼虫的前丝腺在体外显示了对20E的基因反应。体内和体外结果表明异二聚体EcR-B1和USP-2可能是导致细胞死亡的原因。结果还表明E74、E75、BHR3和BR-C亚型参与其中，但与Ftz-F1无关。我们尚未成功地克隆了假定的膜蜕皮激素受体的基因。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。Ca<2+>作为第二信使，mEcR被认为是与Gαq相关的g蛋白偶联受体（GPCR），随后可能通过IP3受体激活磷脂酶c-β，生成肌醇3-磷酸（IP_3），并从内质网释放Ca<2+>，然后Ca<2+>激活蛋白激酶c （PKC）和caspase 3样蛋白酶。这一信号通路最终导致核碎裂和核碎裂。核凝聚是由钙调素和钙调素依赖性蛋白激酶II （CaMK-II）的不同途径调控的。但该通路未被Ca<2+>激活，因此Gαq是否参与该通路尚不清楚。此外，钙调素和CaMK-II抑制剂影响细胞核和DNA片段的发生，表明caspase 3样蛋白酶的激活并不仅仅依赖于GPCR/PLC-β/IP3/Ca<2+>/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.