李芳仁臺灣大學:分子醫學研究所林靜慈Lin, Chin-TszChin-TszLin2010-05-042018-07-092010-05-042018-07-092008U0001-3007200816193500http://ntur.lib.ntu.edu.tw//handle/246246/178687四D腺嘌呤核苷二磷酸核醣化因子相似蛋白 (ARL4D) 隸屬於Ras小分子G蛋白家族中，腺嘌呤核苷二磷酸核醣化因子 (ARF) 次家族成員之一。ARL4D蛋白質表現受不同階段的發育時期調控。近來發現ARL4D作用在cytohesin-2 / ARNO上游；後者為ARF 的鳥糞嘌呤核苷酸轉換因子 (guanine nucleotide exchange factor, GEF)，可刺激ARF6活化並促使肌動蛋白 (actin) 重組和細胞膜的褶皺。在此，我們指出ARL4D可與 Adherens junctions (AJs) 中的重要成員，α-catenin結合。ARL4D在活化態時可與α-catenin胺基酸片段 266至657結合，而非活化態則否。於狗的腎臟上皮細胞株 (Madin-Darby canine kidney, MDCK epithelial cells) 大量表現 ARL4D野生型 (wild type) 或其活化型突變蛋白 (putative active form, ARL4DQ80L)，皆可影響側邊細胞膜 (lateral membranes) 形狀，形成不完整垂直排列與外旋分布，進而造成AJs結構受損。當細胞大量表現ARL4D非活化型突變蛋白 (ARL4DT35N, a putative GTP-binding defective mutant)，則無上述現象發生。 利用Tet-off system篩選受Doxycyclin調控之ARL4D表現細胞株，亦可發現上述現象。綜合以上結果，我們推論ARL4D藉由影響α-catenin與AJs結合的穩定性，進而影響AJs結構。ARL4D is a developmentally regulated protein which belongs to ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related small G proteins. Recently we demonstrated that ARL4D acts as a novel upstream regulator of a guanine nucleotide-exchange factor (GEF), cytohesin-2/ARNO, to promote ARF6 activation and modulate actin remodeling. Here we show that ARL4D interacts with α-catenin, an essential component of adherens junctions (AJs). The residues 266-657 of α-catenin interact with ARL4D in a GTP-dependent manner. Overexpressing ARL4D and its putative active form, ARL4D(Q80L) caused lateral membranes disorganized in Madin-Darby canine kidney (MDCK) epithelial cells. As a consequence, the AJs structure was defective. The lateral membranes appeared less vertical and had convoluted edges in the cells expressing ARL4D and ARL4D(Q80L), but not ARL4D(T35N), a putative GTP-binding defective mutant. Together, our findings suggest that ARL4D affects the stabilization of α–catenin at the cell cortex and alters the structure of AJs complex.Table of content …………………………………………………………………….. 1文摘要 ……………………………………………………………………………. 2bstract ……………………………………………………………………………... 3bbreviations ……………………………………………………………………...... 4ntroduction ………………………………………………………………………… 5aterials and methods ……………………………………………………………. 12esultsubcellular localization of ARL4D and its mutants in MDCK-T23 cells ……… 16-catenin interacted with ARL4D in a GTP-dependent manner ……………….. 16he residues 266-657 of α-catenin were required for ARL4D binding ………... 17ffects of ARL4D on the subcellular localization of α-catenin ………………... 17ffects of ARL4D on the subcellular localization of E-cadherin ……………… 17he expression of ARL4D in MDCK-T23 was doxycyclin-dependent ……….. 18xpression of ARL4D caused a defect in adherens junction structure ………… 18riton X-100 extraction assay ………………………………………………….. 19ther ARL4D(Q80L) expressing cells in MDCK-T23 ………………………… 19RF6(Q67L) induced E-cadherin endocytosis to the perinuclear site in MDCK cells, but ARL4D(Q80L) did not …………………………………………….. 20iscussion ………………………………………………………………………….. 21igure legend ………………………………………………………………………. 23ables ………………………………………………………………………………. 27igures ……………………………………………………………………………... 30eferences …………………………………………………………………………. 49application/pdf17032659 bytesapplication/pdfen-US四D腺嘌呤核苷二磷酸核糖化相似因子ARF-like proteinsARL4Dhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178687/1/ntu-97-R94448010-1.pdf