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RhoD is a member of the classical Rho GTPases and it has essential roles in the regulation of actin dynamics. RhoD localizes to early endosomes and recycling endosomes, which indicates its important role in the regulation of endosome trafficking. Here, we show that RhoD binds to the Rab5 effector Rabankyrin-5, and RhoD and Rabankyrin-5 colocalize to Rab5-positive endosomes, which suggests a role for Rabankyrin-5 in the coordination of RhoD and Rab5 in endosomal trafficking. Interestingly, depletion of RhoD using siRNA techniques interfered with the internalization of the PDGFβ receptor and the subsequent activation of the downstream signaling cascades. Our data suggest that RhoD and Rabankyrin-5 have important roles in coordinating RhoD and Rab activities during internalization and trafficking of activated tyrosine kinase receptors.
Vital cellular processes such as intracellular trafficking of organelles and vesicles, cell migration and signal transduction require the precise orchestration of the mechanisms that govern cytoskeletal and membrane dynamics . Members of the Rho GTPases are key elements in the machinery that regulates cytoskeletal dynamics, and thereby the signaling pathways that regulate the ability of cells to respond and adapt to the extracellular environment . In humans, the Rho subfamily of small GTPases includes 20 proteins . Despite the large number of Rho GTPases, the vast majority of studies still focus on the three classical Rho GTPases: Cdc42, Rac1 and RhoA.
In addition to being key regulators of cytoskeletal reorganization, some of the small GTPases have additional roles in the regulation of membrane dynamics. For instance, Cdc42 is involved in the regulation of polarized transport of vesicles, and thereby, is involved in the establishment of apical to basal polarity in epithelial cells [3, 4]. Studies on RhoB implicate it in the control of the endocytic pathway. Depending on the cellular context, RhoB can facilitate trafficking of signaling molecules, including receptor-tyrosine kinases, Akt and Src, to the cell surface, the nucleus or lysosomes [5-7]. Another key operator in this context is the less-studied GTPase RhoD, as it has been shown to have important roles in coordinating actin reorganization and endosomal trafficking [8, 9].
RhoD was first identified by Chavrier et al. using a polymerase chain reaction (PCR) cloning approach . Together with the related GTPase Rif, it constitutes a unique subgroup of the classical Rho GTPases [8-13]. Both RhoD and Rif have profound effects on the organization of the actin filament system. Ectopic expression of active variants of RhoD and Rif in HeLa cells and baby hamster kidney cells promotes the formation of long protrusions that emerge from the periphery or from the dorsal side of the cells [8, 12]. In addition, RhoD and Rif have been shown to trigger the formation of peripheral protrusions in porcine aortic endothelial cells . Ectopically expressed RhoD and Rif localize to the plasma membrane, and RhoD has also been seen to localize to vesicles throughout the cytoplasm [8, 12, 15, 16]. These vesicles are positive for Rab5, which implies that they represent early endosomes. There are indications of a collaboration between Rab5 and RhoD in the regulation of endosome movement, as the enlargement and perinuclear accumulation of endosomes induced by overexpression of a constitutively active Rab5 (Rab/Q79L) can be antagonized by simultaneous expression of a constitutively active RhoD mutant (RhoD/G26V) .
Clearly, RhoD has a role in the regulation of cell migration, as activation of RhoD has been negatively correlated with cell locomotion in several assays [15, 17]. To date, the very few RhoD-binding partners that have been identified include the diaphanous-related formins, hDia2 and mDia1 and the Semaphorin receptors PlexinB1 and PlexinA1 [16, 18-21]. However, these RhoD-binding proteins do not provide clues to the mechanisms underlying the diverse cellular effects of RhoD. Therefore, we performed yeast two-hybrid screening to find out more about the mechanisms underlying RhoD-dependent effects on actin reorganization and membrane trafficking. We identified the Rab5 effector Rabankyrin-5 as a RhoD-binding protein. In the same screen, we identified FilaminA-interacting protein 1 (FILIP1), the actin nucleation-promoting factor WASP homolog associated with actin Golgi membranes and microtubules (WHAMM), and zipper-interacting protein kinase (ZIPK) [22, 23]. Rabankyrin-5 has been shown to regulate macropinocytosis . Our data suggest that Rabankyrin-5 has an important function in the coordination of Rab5- and RhoD-regulated cellular processes. In particular, we show that RhoD and Rabankyrin-5 participate in the regulation of the internalization of platelet-derived growth factor-β receptor (PDGFRβ). This implies a novel function of RhoD in the regulation of receptor tyrosine kinase trafficking.
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Several lines of evidences implicate RhoD as a central player in orchestrating signaling networks that integrate cytoskeletal dynamics and membrane trafficking. However, the molecular mechanisms underlying this integration are unknown. Our studies indicate that RhoD can recruit specific downstream binding partners and thereby regulate different processes. Additionally, RhoD is likely to cooperate with other small GTPases in the control of endosomal trafficking. For instance, overexpression of constitutively active Rab5 has been shown to lead to increased endosomal vesicle movement, as well as vesicle fusion, which can result in the formation of large perinuclear endosomes [1, 8]. Expression of constitutively active RhoD inhibited this Rab5-dependent effect and resulted in the formation of smaller, more spherical and scattered endosomes . This RhoD-dependent effect on endosome trafficking was also observed independently of Rab5 overexpression, which indicates that RhoD activity alone is required for the disturbed endosomal movement.
Rabankyrin-5 is not the only RhoD-binding protein with an identified role in endosomal trafficking. RhoD has been shown to bind and relocalize the Diaphanous-related formin (DRF) hDia2 to endosomal membranes, and hDia2 is required for the RhoD-dependent block of the endosomal movement. Overexpression of RhoD and hDia2 also promotes the relocalization of the non-receptor tyrosine kinase c-Src to endosomal vesicles, along with potent stimulation of c-Src activity . RhoD-positive endosomes were shown to have a striking alignment along actin filaments, which suggests that the block in endosome movement is due to an increased association of the vesicles to actin filaments . It is likely that DRFs, such as hDia2C, can collaborate with Rabankyrin-5 in the control of endosomal trafficking.
The identification of Rabankyrin-5 as a RhoD-binding partner provides a novel and important clue to the role of RhoD in endosomal trafficking. Rabankyrin-5 has been shown to localize to early endosomes and to apical macropinosomes in epithelial cells . Studies by Schnatwinkel et al.  showed that transient transfection of Rabankyrin-5 in NIH3T3 fibroblasts and in MDCK epithelial cells resulted in an increased number of macropinosomes and increased fluid-phase uptake. In contrast, knock-down of Rabankyrin-5 reduced these processes.
RhoD has a profound effect on the organization of the actin filament system [8, 14]. The mechanisms underlying this control are not clear, but RhoD has been shown to interact with the DRF mDia1, which is known to be a key denominator in the regulation of actin polymerization [18, 27]. Surprisingly, the main role of mDia1 downstream of RhoD appears to be linked to the regulation of cell-cycle progression and centrosome duplication, rather than to the regulation of actin dynamics . Previously, we observed that RhoD binds to the actin nucleation-promoting factor WHAMM and the FilaminA-interacting protein FILIP1. Together with these binding partners, RhoD forms a signaling pathway that regulates cell attachment and cell migration .
There is clear correlation between endocytosis and cell signaling . The controlled internalization of transmembrane receptors constitutes an important step in the regulation of the activity of these receptors. There is also crosstalk between the endocytosis machinery and the apparatus that regulates cell migration . In addition to RhoD, a few other members of the Rho GTPases have been shown to contribute to the control of membrane trafficking. For instance, Rac1 was shown to stimulate membrane ruffling as well as micropinocytosis . Cdc42 has a role in the regulation of polarized transport of vesicles and also in the establishment of apical to basal polarity in epithelial cells [3, 4]. Moreover, the atypical Rho GTPase RhoBTB2 has been proposed to have a role in endocytosis . In addition, RhoB has been shown to regulate the endosomal targeting of receptor tyrosine kinases, such as EGF and PDGFRβ [7, 31-33].
Interestingly, RhoB has been shown to work in consort with RhoD in membrane targeting and activation of the non-receptor tyrosine kinases Src, Yes and Fyn . We found that knock-down of RhoD, and to some extent of Rabankyrin-5, resulted in defective PDGFRβ internalization and the subsequent decrease in the activation of PDGFRβ. Importantly, we observed mutual dependency of RhoD and Rabankyrin-5, as the knock-down of either of these two proteins resulted in markedly disturbed subcellular localization of the other of these proteins. This suggests that RhoD and Rabankyrin-5 can, at least to a certain extent, be hypothesized to act in the same pathway in endosomal positioning, but that they function distinctly from each other in their regulation of PDGFRβ dynamics.
Several binding partners of the Rho GTPases have dual roles in actin regulation and membrane dynamics, such as the WASP family proteins and the F-BAR proteins . Previously, we have shown that knocking down the CIP4 family of F-BAR proteins results in decreased internalization of PDGFRβ . In this study, we show that the prolonged cell-surface exposure of PDGFRβ results in increased receptor activation, as well as in an increased and sustained formation of PDGF-BB-induced dorsal ruffles in fibroblasts . RhoD knock-down appears to have the opposite effects on receptor activation, indicating that the underlying molecular mechanisms are different. It is clear that membrane trafficking and cytoskeletal dynamics are processes that need to be coordinated. RhoD constitutes an attractive candidate as a master regulator for the integration of these processes.
Our studies demonstrate that RhoD and Rabankyrin-5 have a critical role in the control of endocytosis. The interaction between Rabankyrin-5 and RhoD is independent of the GTP-loaded status of RhoD. The localization of RhoD to early endosomes is dependent on the membrane targeting CAAX box, rather than on its GTP-loaded status, whereas GDP-loaded Rab5 does not localize to early endosomes. Interestingly, recent observations indicate that RhoD is regulated in yet another fashion compared to the classical Rho GTPases, as its intrinsic nucleotide exchange activity is much greater . Thus, it is unlikely that RhoD is regulated by GEFs and GAPs, which is in contrast to the classical Rho members RhoA, Rac1 and Cdc42. Rabankyrin-5 might function as a RhoD regulator, by sequestration of RhoD, and the consequent modulation of its activity. Furthermore, it is probable that Rabankyrin-5 communicates with other RhoD-binding partners, such as WHAMM, in the control of vital cellular processes, such as cell migration, although this remains to be defined pending further studies.