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

Functional characterization of human ARF-like proteins, ARL4D and its interacted protein α-catenin

林靜慈(Chin-Tsz Lin)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.03170

摘要

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。

關鍵字

四D腺嘌呤核苷二磷酸核糖化相似因子

並列摘要

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.

並列關鍵字

ARF-like proteins ； ARL4D

參考文獻

Altschuler, Y. et al. ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells. J Cell Biol 1999, 147, 7–12.

Bhamidipati, A., Lewis, S. A. and Cowan, N. J. ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin. J Cell Biol 2000, 149, 1087-1096.

Bullions, L. C., Notterman, D. A., Chung, L. S. and Levine, A. J. Expression of wild-type α-catenin protein in cells with a mutant α-catenin gene restores both growth regulation and tumor suppressor activities. Mol Cell Biol 1997, 17, 4501–4508.

Chavrier, P. and B. Goud. The role of Arf and Rab GTPases in membrane transport. Curr Opin Cell Biol 1999, 11, 466-475.

D’Souza-Schorey, C. Disassembling adherens junctions: breaking up is hard to do. Trends Cell Biol 2005, 15, 19-26.