ZNRF1在表皮生長因子受體的運輸與降解所扮演之角色

The role of ZNRF1 in EGFR trafficking and degradation

陳彥剴(Yen-Kai Chen)

指導教授 ： 徐立中

國立臺灣大學/醫學院/分子醫學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602044

摘要

表皮生長因子受體(EGFR)是一種受體酪胺酸激酶(RTK)，並且參與在許多細胞代謝的過程當中，包括細胞的增生、分化與存活。表皮生長因子受體在細胞當中表現量的異常，以及訊息傳遞過度的活化會導致許多的疾病，例如：癌症；所以準確的調控表皮生長因子受體在細胞內的運輸與降解是相當重要的。有許多的證據指出表皮生長因子受體的磷酸化(phosphorylation)與泛素化(ubiquitination)會和它的運輸與降解的過程有關係，但是詳細的分子機制到目前為止還不清楚。最近我們發現了一個E3泛素接合酶ZNRF1蛋白，它可以在肺癌細胞當中去調控表皮生長因子受體的降解，因此我們想要進一步去探討ZNRF1是如何去調控表皮生長因子受體的運輸與降解。首先，我們在非小細胞肺癌細胞株A549中利用CRISPR/ Cas9的技術剔除ZNRF1基因，然後在這樣的細胞當中加入會促使表皮生長因子受體開始降解的表皮生長因子配體(EGF)，我們發現表皮生長因子受體的降解會因此而減緩。然而，在這樣的細胞當中重新表現野生型(wild-type)的ZNRF1會使得表皮生長因子受體原本減緩的降解速率得到回復，但如果是表現酵素活性受到突變的ZNRF1則無法達到回復的效果；這樣的結果顯示ZNRF1的酵素活性對於調控表皮生長因子受體的降解是必須的。此外，我們發現ZNRF1會與表皮生長因子受體的酪胺酸激酶結構區(tyrosine kinase domain; TKD)有交互作用，並且會調控受體的泛素化。接著，我們發現了第五號銜接蛋白複合體(AP-5)的其中一個成員SPG11也會參與在表皮生長因子受體的降解過程，因為當我們在肺癌細胞中降低SPG11蛋白表現量的時候，同樣會減緩表皮生長因子受體的降解。雖然我們還需要許多的研究才能更全面性的了解ZNRF1調控表皮生長因子受體運輸和降解的機制；但總體而言，我們的實驗結果指出ZNRF1會與表皮生長因子受體(EGFR)產生交互作用，並且可能是藉由調控第五號銜接蛋白複合體(AP-5)來協助受體的運輸與降解，以達到調控表皮生長因子受體降解與訊息傳遞的平衡。

關鍵字

表皮生長因子受體 ； 表皮生長因子配體 ； 受體酪胺酸激? ； 泛素化

並列摘要

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that plays roles in numerous cellular processes, including cell proliferation, survival and differentiation. Downregulation or overactivation of EGFR has been associated with many human diseases, such as cancer. Endosomal trafficking and degradation of EGFR is crucial for controlling its downstream signaling. Mounting evidence suggests that ubiquitination and phosphorylation of EGFR are important for its trafficking and degradation, but the molecular mechanism remains unclear. We recently found an E3 ubiquitin ligase, ZNRF1, mediate EGFR degradation in lung cancer cell lines. Here, we aim to elucidate how ZNRF1 mediates EGFR trafficking and degradation. We first generated ZNRF1-/- A549 cells using the CRISPR/Cas9 system, and confirmed that EGF-triggered EGFR degradation was decreased in these cells. Reconstitution of wild-type, but not the catalytically inactive E3 ligase, ZNRF1 to ZNRF1-deleted cells restored the effect of ZNRF1-mediated EGFR degradation, suggesting a requirement of the E3 ubiquitin ligase in the ZNRF1-mediated EGFR trafficking. In addition, we found that ZNRF1 physically associated with the tyrosine kinase domain (TKD) of EGFR and regulated its ubiquitination. Depletion of SPG11, a component of the adaptor protein-5 (AP-5) complex, in A549 cells decreased ligand-induced EGFR degradation, suggesting also its involvement in EGFR degradation. Together, our data suggest that ZNRF1 interacts and controls EGFR trafficking and degradation possible through the AP-5 complex. Nevertheless, more studies are needed to completely understand the underlying mechanism by which ZNRF1 regulates EGFR trafficking and degradation.

並列關鍵字

ZNRF1 ； EGFR ； EGF ； RTK ； ubiquitination

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