Characterization of the roles of Rab5 isoforms in endocytic membrane traffic and cell migration.

摘要

Rab5 isoforms (A, B and C) share ∼ 80 % of sequence identity. Work from several labs indicates that the three Rab5 isoforms are ubiquitously expressed but transcriptionally regulated and phosphorylated, suggesting that they may play different physiological roles. We took advantage of the RNAi-silencing technique to individually silence endogenous isoforms in HeLa cell line, so that we could examine their loss-of-function phenotypes more likely to capture their physiological activities.;In this thesis, I demonstrated that suppression of Rab5A or 5B delays the degradation of EGFR in HeLa and DU145 cells, and impairs the exit of EGFR from early endosomal compartment; whereas Rab5C depletion has very little effect. We examined the endogenous abundance of each Rab5 isoform and determined that Rab5A is no more enriched that other isoforms in HeLa or DU145 cells. Therefore, the more robust effect on EGFR degradation in the absence of Rab5A is due to its specificity in this pathway, not its abundance. Furthermore, HeLa cells depleted of different isoforms showed distinct morphological alteration, suggesting a unique correlation between the isoforms and cytoskeletal rearrangement. By means of monitoring the activation of Rac and wound closure assay, we found that Rab5C depletion has more impact on Rac activity and cell motility. In the mean time, we investigated the specificity of Rab5 exchange factors in regulating Rab5 isoform-associated functions, and found that EGF-induced Rab5 isoform activation and Rab5 isoform-induced Rac activation are differentially altered by distinct exchange factors. Additionally, examination of the binding specificity of Rab5 exchange factors, Rin1 and Rin3, and Rab5 subgroup members showed that Rin1 preferentially binds to Rab5A. On the other hand, Rin3 preferentially interacts with Rab22A, which activates Rab22A and tubulates Rab22A vesicles. Taken together, the study presented in this thesis provides new insight into the functional significance of Rab5 isoforms, and the differential regulation provided by their exchange factors.