Functional characterization of human Arl4D and its interacting protein, IRSp53/BAIAP2
| Autor: | Tai-Chen Tsai, 蔡岱蓁 |
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| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 ADP-ribosylation factors (Arfs) are small GTP binding proteins involved in membrane transport, the maintenance of organelle integrity and cytoskeletal dynamics. The Arf-like 4 (Arl4) family is composed of three isoforms, Arl4A, Arl4C and Arl4D. Here, we identified a novel interacting protein of Arl4D, called insulin substrate receptor p53 (IRSp53). IRSp53 is a main actin modulator that regulates filopodia and lamellipodia formation, mediated by two small GTPases, Cdc42 and Rac1, respectively. Through yeast two hybrid, we found that only Arl4D, but not Arl4A or Arl4C, interacted with IRSp53. Confirmed by in vitro binding assay and co-immunoprecipitation, we demonstrated that both constitutively active and inactive mutants of Arl4D interacted with IRSp53. Revealed from yeast two hybrid and in vitro binding assay, the shortest region important for their interaction resided in the N terminal 229 residues of IRSp53, which is the same as reported Rac1 binding region. Knockdown of IRSp53 did not affect the membrane localization of Arl4D. By contrast, knockout of Arl4D decreased the number of IRSp53-induced filopodium-like protrusions. In addition, coexpression of WT and constitutively active but not inactive mutant of Arl4D reduced the average length of IRSp53-induced protrusions. Last but not least, Arl4D was found within the same complex with Cdc42 and IRSp53 from co-immunoprecipitation. In conclusion, we found that Arl4D is a novel small GTPase that interacts with IRSp53, which might regulate filopodia formation through unknown mechanism via IRSp53. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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