Abstract The tyrosine kinase activity and endocytosis of epidermal growth factor receptor (EGFR) are activated by ligand binding. The adaptor protein 2 (AP2) complex found in the clathrin coated vesicles mediates intracellular trafficking of EGFR. Accumulating evidence suggest that the nucleus is one of intracellular destination. Nuclear EGFR severs as a transcriptional co-activator to regulate expression of several genes which are involved in tumor growth, progression and drug resistance. The carboxyl terminal 186 amino acids of EGFR has transcriptional activation activity and the AT-rich sequence (ATRS) of the target gene promoter is required for EGFR binding. In the present study, we show that AP2A1, the alpha 1 adaptin subunit of AP2 complex, interact with EGFR not only in the cytoplasm but also in the nucleus. The EGFR-AP2A1 interaction is stimulated by EGF and suppressed by Iressa, a tyrosine kinase inhibitor. Knockdown AP2A1 expression in cancer cells abrogates EGF-induced EGFR nuclear translocation. Interesting, the ATRS reporter activity is enhanced by depletion of cellular AP2A1. These results indicate that AP2A1 is required for EGFR nuclear translocation and suppresses the transcriptional activation activity of EGFR. Thus, we demonstrate here the novel role of AP2A1 in EGFR trafficking and transcriptional regulation. Citation Format: Huai-Gu Chen, Sheng-Chieh Hsu. Role of AP2A1 in EGFR nuclear translocation and transcriptional activation activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2014-3325