人類四D腺嘌呤核苷二磷酸核糖化相似因子與其結合蛋白α-catenin之特性探討

Abstract

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.