Rabex-5 Protein Regulates the Endocytic Trafficking Pathway of Ubiquitinated Neural Cell Adhesion Molecule L1*

Background: The endocytic trafficking pathway for L1 proteolytic degradation following L1-L1 homophilic binding remains unclear. Results: Changes in Rabex-5 expression alter the dynamics of ubiquitinated L1 endocytic trafficking. Conclusion: Rabex-5 has an essential role in the endocytic trafficking of ubiquitinated L1. Significance: The motif interacting with ubiquitin (MIU) domain, not the A20 ZnF domain, in Rabex-5 controls the endocytic trafficking of ubiquitinated cargo. Ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of cell surface proteins in eukaryotic cells. Ubiquitin (Ub)-binding proteins (UBPs) regulate the stability, function, and localization of ubiquitinated cell surface proteins in the endocytic pathway. Here, I report that the immunoglobulin superfamily cell adhesion molecule L1 undergoes ubiquitination and dephosphorylation on the plasma membrane upon L1 antibody-induced clustering, which mimics L1-L1 homophilic binding, and that these modifications are critical for obtaining the maximal rate of internalization and trafficking to the lysosome, but not to the proteasome. Notably, L1 antibody-induced clustering leads to the association of ubiquitinated L1 with Rabex-5, a UBP and guanine nucleotide exchange factor for Rab5, via interaction with the motif interacting with Ub (MIU) domain, but not the A20-type zinc finger domain. This interaction specifically depends on the presence of an Ub moiety on lysine residues in L1. Rabex-5 expression accelerates the internalization rates of L1WT and L1Y1176A, a tyrosine-based motif mutant, but not L1K11R, an ubiquitination-deficient mutant, leading to the accumulation of ubiquitinated L1 on endosomes. In contrast, RNA interference-mediated knockdown of Rabex-5 impairs the internalizations of L1WT and L1Y1176A, but not L1K11R from the plasma membrane. Overall, these results provide a novel mechanistic insight into how Rabex-5 regulates internalization and postendocytic trafficking of ubiquitinated L1 destined for lysosomal degradation.

L1 is an immunoglobulin superfamily cell adhesion molecule implicated in a number of developmentally important processes, including neuronal cell migration, axon outgrowth, and axon fasciculation (1,2). It is well established that endocytic retrieval from the rear of the growth cone maintains an L1 gradient across the growth cone and provides a pool of vesicular L1 available for local exocytosis (3)(4)(5). The endosomal trafficking is also required to target L1 properly to the growing axon. L1/neuron-glia cell adhesion molecule reaches the axon indirectly by transcytosis, involving initial somatodendritic targeting followed by endocytosis and trafficking to the axon from somatodendritic endosomes (6 -8). L1 endocytosis is substrate-dependent process and occurs as a consequence of L1-L1 homophilic binding. The cytoplasmic tail of L1 contains a tyrosine-based recognition motif (YRSLE) that mediates binding to the AP-2 clathrin endocytosis adaptor (5). L1-L1 homophilic binding causes dephosphorylation of this tyrosine-based motif and triggers endocytosis via recruitment of AP-2 (9). In contrast, binding to AP-2 is reduced upon phosphorylation of the tyrosine-based motif downstream of Src signaling (9). Although the dynamic nature of L1-L1 homophilic binding has been studied in detail by using sophisticated live imaging and quantitative tracking approaches (10), the molecular components of postendocytic delivery to lysosomes for down-regulation upon L1-L1 homophilic binding are yet to be identified and characterized.
Posttranslational attachment of ubiquitin (Ub) 2 to polypeptides has a fundamental role in modulating the plasma membrane protein composition (11)(12)(13). The current paradigm (supported by studies on chimeric receptor-Ub fusion proteins in yeast and mammalian cells) suggests that ubiquitination promotes the internalization and sorting of cargo receptors by recruiting Ub-binding proteins (UBPs), which link cargo to components of endocytic and sorting machinery (14 -16). A number of UBPs, including Rabex-5, present at endosomes are able to participate in degradative sorting of ubiquitinated cargo (14 -16). Rabex-5, originally identified as a rabaptin-5-interacting protein, possesses GEF activity for Rab5 (17). It is a multidomain protein consisting of an A20-type zinc finger (ZnF) fused to a motif interacting with Ub (MIU) domain at the N * This work was supported by Grant-in-aid for Young Scientists (B) terminus, membrane-binding motif, and tandem helical bundle-VPS9 domains at the center, and a coiled coil region at the C terminus. Rabex-5 binds Ub through the tandem ZnF and MIU UBDs and undergoes UBD-coupled monoubiquitination driven by ZnF Ub-ligase activity (18,19). Rabex-5 GEF activity is mediated by the helical bundle-VPS9 tandem domains (20). The coiled coil region of Rabex-5 forms a complex with rabaptin-5 and indirectly targets Rabex-5 to early endosomes via rabaptin-5 binding to Rab5-GTP (21,22). The multidomain architecture of Rabex-5 allows it to perform dual roles in cargo sorting by simultaneously binding to the Ub-binding and ubiquitinated adaptors and promoting Rab5-dependent endosomal fusion.
In this study, I investigated Rabex-5 function in the regulation of internalization, sorting, and lysosomal degradation of ubiquitinated L1 upon L1-L1 homophilic binding. I report that incubation with an L1 antibody (L1-Ab) that mimics L1-L1 homophilic binding leads to ubiquitination and dephosphorylation at the tyrosine-based motif, which facilitates endocytosis via recruitment of Rabex-5. Interfering with L1 ubiquitination reduces L1 internalization due to impairment of the interaction with Rabex-5. It is, therefore, plausible that Rabex-5 plays an important role in neuronal function by controlling L1 internalization and endocytic sorting.
Cell Culture and cDNA Transfection-Mouse neuroblastoma N2a cells and human embryonic kidney 293T (HEK293T) cells were maintained in DMEM supplemented with 10% heatinactivated fetal bovine serum (FBS). Plasmid DNA was transfected into cells by using Lipofectamine 2000 (Invitrogen).
Subcellular Fractionation-Cells were rinsed with ice-cold PBS and scraped into fractionation buffer (100 mM Tris-HCl, pH 7.4, containing protease inhibitors). Cells were then homogenized using a Dounce homogenizer and centrifuged at 850 ϫ g for 10 min to remove nuclei and cell debris, and postnuclear supernatants were subjected to ultracentrifugation at 200,000 ϫ g for 10 min in a Himac CS120GXL centrifuge (Hitachi, Tokyo, Japan) to separate the membrane (pellet) and cytosolic (supernatant) fractions. The pellet was resuspended in fractionation buffer. Proteins in each fraction (50 g/l) were analyzed by SDS-PAGE and immunoblot assay, as described above.
Biotinylation Assay for Endocytosis-Cells pretreated with cycloheximide (CHX; 10 g/ml) and leupeptin (0.3 mM) were washed with ice-cold PBS and biotinylated by incubating with 300 g/ml EZ-Link-Sulfo-NHS-SS-Biotin (Pierce) for 30 min at 4°C. Excess biotin was quenched by washing with DMEM. Following this, DMEM at 37°C was added, and biotinylated cells were treated with polyclonal L1-Ab for the indicated times. Remaining cell surface biotin was stripped using stripping solution (50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, and 1% FBS). Cell extracts were made, and cell debris was removed by centrifugation at 14,000 ϫ g for 20 min. Clarified cell extracts were precipitated using streptavidin and immobilized on agarose beads at 4°C for 2 h. After washing five times with cell lysis buffer, the bound proteins were removed with SDS sample buffer.
Imaging and Quantification-After transfection (48 h), cells were rinsed with PBS, fixed in 4% formaldehyde for 30 min, and permeabilized with 0.3% Triton-X in PBS for 30 min. Primary antibodies were diluted in PBS containing 10% FBS. Labeled cells were visualized using a 1X71 fluorescence microscope (Olympus, Tokyo, Japan) with a 60ϫ oil immersion objective lens. Quantification of surface and/or intracellular fluorescence intensities of L1 was done with MetaMorph imaging software (Universal Imaging Corp.) using an arbitrary threshold. To examine colocalization of fluorescence signals in different channels, the MetaMorph colocalization function following background subtraction and threshold setting were used. Laser-scanning confocal microscopy was performed using an Olympus FV-1000 equipped with a 63ϫ oil immersion objective lens. In at least three independent experiments, 30 cells were photographed and analyzed for each construct. Statistical analysis was done using ANOVA and post hoc tests with appropriate Bonferroni adjustment for multiple comparisons, to ensure a significance level of 0.05 in all experiments. *, **, and *** represent Ͻ0.05, Ͻ0.01, and Ͻ0.001, respectively. Error bars denote the S.E.
Next, I asked whether L1-Ab-dependent internalization of L1 is involved in triggering posttranslational modification of L1, such as dephosphorylation of the tyrosine-based motif (9) and ubiquitination which is critical role in sorting of NCAM, DM-GRASP, and L1 (25)(26)(27). Consistent with previous results (9,25), L1-Ab treatment led to L1 monoubiquitination ( To examine whether phosphorylation of the tyrosine-based motif is controlled by the Src kinase, as reported previously (9), the cells were pretreated with the Src kinase inhibitor PP2 and then incubated with L1-Ab. As expected, dephosphorylation of the L1 tyrosine-based motif in PP2-pretreated cells significantly increased by 1.5 Ϯ 0.1-fold compared with control cells ( Although the apparent dependence of endocytosis on ubiquitination suggests an early role for the ubiquitination process in internalization, it is possible that L1 ubiquitination occurs after internalization and that its role may instead be to prevent receptor recycling to the plasma membrane from an internalized pool. To address this question, I inhibited the progression of endocytosis by expressing dominant negative dynamin, namely dynamin K44A (28). Expression of dynamin K44A drastically inhibited L1 internalization after L1-Ab incubation compared with control cells (Fig. 2D, middle panels). Importantly, compared with the nontransfected cells, L1 ubiquitination in cells expressing dynamin K44A significantly increased before and after L1-Ab incubation by 2.2 Ϯ 0.4-fold (Fig. 2D, lower panels, lane 3 versus lane 1, n ϭ 3, p Ͻ 0.01) and 1.4 Ϯ 0.2-fold (Fig. 2D, lower panels, lane 4 versus lane 2, n ϭ 3, p Ͻ 0.01), respectively, suggesting that ubiquitination occurs before internalization from the plasma membrane.

Rabex-5-dependent Sorting of Ubiquitinated L1
To address the relationship between L1 ubiquitination and protein degradation, I measured the metabolic stability of endogenous L1 by immunoblotting following inhibition of protein synthesis with CHX. In the presence of L1-Ab, L1 had a significantly faster turnover rate than that in the absence of L1-Ab, its half-life being 4.3 h and 1.9 h in the absence and presence of L1-Ab, respectively (Fig. 2E). The accelerated degradation of L1 in the presence of L1-Ab can at least in part be attributed to lysosomal proteolysis because preventing lysosomal acidification and delivery by using bafilomycin A1, a vacuolar H ϩ -ATPase inhibitor, significantly delayed L1 degradation (Fig. 2E). On the contrary, the cysteine protease and proteasomal inhibitor lactacystin partially stabilized L1 (Fig.  2E). Overall, these results suggest that L1 is subjected to both mono-and polyubiquitination on the plasma membrane and is sorted into lysosomes, but not proteasomes, via Rab5 early endocytic trafficking pathway upon L1-L1 homophilic binding.
Ubiquitination Plays an Important Role in L1 Internalization and Targeting to Lysosomal Compartments-To establish the role of L1 Ub modification on internalization and traffic to the lysosome, I constructed a ubiquitination-deficient mutant of L1, L1 K11R , in which a Lys is replaced with Arg at position 11 in the cytoplasmic tail (Fig. 3A). The intracellular domain of L1 contains 11 Lys residues, two of which form a putative ezrinbinding motif (29) (Fig. 3A). As expected, ubiquitination of L1 K11R mutants was drastically reduced by 80 -90% compared with L1 WT (Fig. 3B). Intriguingly, Myc-tagged ezrin did not coimmunoprecipitate with FLAG-tagged L1 in the cells overexpressing HA-tagged Ub (Fig. 3C) but did in control cells, providing the possibility that ubiquitination within the L1 ezrinbinding site interferes with ezrin binding because of steric hindrance by ubiquitination.
Because the cytoplasmic domain of L1 is known to contain a tyrosine-based sorting motif, I constructed a single amino acid substitution of Tyr 1176 to Ala within this motif to compare the

Rabex-5-dependent Sorting of Ubiquitinated L1
rate of internalization of this mutant with L1 K11R . Consistent with the enhancement of L1 ubiquitination by PP2 (Fig. 2C,  right panel), the extent of L1 Y1176A ubiquitination significantly increased by 1.3 Ϯ 0.1-fold compared with the L1 WT (Fig. 3B, lane 4 versus lane 2, n ϭ 3, p Ͻ 0.01), suggesting that dephosphorylation at the tyrosine-based motif plays a critical role in the ubiquitination of L1. However, its internalization is grossly retarded after 15-min incubation (16.8% Ϯ 2.4%) and only slightly greater than that of L1 K11R after 30 min (Fig. 3D). These results confirm that ubiquitination is not sufficient for rapid L1 internalization and turnover and suggest that the endocytic motif also plays a key role in this process. I next examined whether L1 Y1176A and L1 K11R are targeted to the endosomes and lysosomal compartments after internalization. L1 Y1176A was detected on Rab5-and Rab11-positive endosomes and LysoTracker-positive lysosomes upon L1-Ab incubation (Fig.  3E, arrows). Although L1 K11R was also present on Rab5-and Rab11-positive endosomes, only a little L1 K11R was detected on LysoTracker-positive lysosomes in the presence of L1-Ab. Image quantification revealed that colocalization of L1 WT , L1 K11R , and L1 Y1176A with lysosomes was 18.7% Ϯ 1.5%, 0.8% Ϯ 0.1%, and 17.2% Ϯ 1.1%, respectively (Fig. 3F), suggesting that ubiquitination is required for the L1 protein to be internalized and targeted for lysosomal degradation.
Rabex-5 Interacts Directly with Ubiquitinated L1 via MIU-As extensive colocalization of endogenous L1 and Rab5 was observed following L1-Ab incubation ( Fig. 2A), I next sought to determine whether Rab5 is involved in L1 internalization by expressing wild-type Rab5, or GTPase-deficient Rab5 Q79L , or GTP-binding-deficient Rab5 S34N mutants in N2a cells. The Rab5 S34N dominant negative GFP-Rab5 mutant (30) did not affect endogenous L1 distribution (Fig. 4A). However, endogenous L1 was significantly redistributed from the plasma membrane to enlarged vacuoles by the expression of the constitutively active Rab5 Q79L mutant (30) in the absence of L1-Ab (Fig. 4A,  arrows), and redistribution was further enhanced in the presence of L1-Ab (data not shown). The impact of Rab5 Q79L on L1 redistribution is similar to its enhancement of the intracellular distribu-tion of the EGF and m4 muscarinic acetylcholine receptors (31,32). Intriguingly, enhanced L1 redistribution in the cells expressing Myc-tagged Rabex-5 was greater than in the cells expressing the GFP-Rab5 Q79L mutant (Fig. 4A). Given that the GTP-bound activated form of Rab5 promotes L1 internalization, I measured the amount of cell surface biotinylated L1 in cells expressing Rab5 Q79L or Rabex-5 before and after L1-Ab incubation. As expected, there was significantly less biotinylated L1 on the surface of cells expressing Rab5 Q79L and a further reduction in cells expressing Rabex-5 before and after L1-Ab incubation (Fig. 4B). Furthermore, Rabex-5 was translocated to the plasma membrane (Fig. 4C, middle panels) and enlarged endosomal compartments upon incubation with L1-Ab (Fig. 4 C, lower panel). Taken together, these data suggest that L1-Ab clustering leads to Rab5 GEF activation via Rabex-5 recruitment to the plasma membrane to accelerate the internalization of L1. Surprisingly, the amount of ubiquitinated L1 was dramatically increased by 6.8 Ϯ 0.9-fold in cells expressing Rabex-5 compared with the untransfected cells (Fig. 4D). However, these results raised the question of why prolonged Rabex-5 expression should cause significant L1 redistribution from the plasma membrane into the endosomes.
Because L1 is ubiquitinated and Rabex-5 contains UBDs, I next examined whether Rabex-5 causes ubiquitinated L1 to accumulate through a direct interaction. Rabex-5 was found to coimmunoprecipitate with L1 in untreated cells, and this was
Because Rabex-5 contains two independent UBDs in the N terminus (18,19), I next examined which of these domains mediates the interaction with ubiquitinated L1. For this, I constructed the Rabex-5 Y25A/A58D double mutant, mutated in the both the A20 ZnF and MIU domains, and the respective single mutants (Fig. 5D). Rabex-5 Y25A , but not Rabex-5 A58D or Rabex-5 Y25A/A58D proteins, coimmunoprecipitated with L1 (Fig. 5D), suggesting that the MIU domain is critical for Rabex-5 association with ubiquitinated L1. Although the A20 ZnF domain exhibits Ub-ligase activity (18,19), knockdown of Rabex-5 with siRNAs did not affect the level of L1 ubiquitination relative to the controls either before or after L1-Ab stimulation (data not shown), indicating that L1 is not a substrate for Rabex-5 Ubligase activity. Consistent with a previous report that membrane targeting of Rabex-5 is mediated by the association of Rabex-5 UBDs with ubiquitinated membrane proteins (33), all three Rabex-5 mutants redistributed from the membrane to the cytosolic fraction (Fig. 5, E and F).
To further establish differences in the functional properties between these UBDs, I compared the amounts of internalized L1 in cells expressing Rabex-5 mutants (Fig. 5E). Rabex-5 WT or Rabex-5 Y25A expression redistributed L1 from the plasma membrane to the endosomes even in the absence of L1-Ab (Fig.  5E, arrows), causing an accumulation of the ubiquitinated L1. In contrast, Rabex-5 A58D and the Rabex-5 Y25A/A58D double mutant had no effect on L1 distribution (Fig. 5E). These results suggest that the MIU domain, but not A20 ZnF, plays an important role in L1 internalization through direct interaction with ubiquitinated L1.

DISCUSSION
Both activation and attenuation of the L1 signal are tightly regulated by endocytosis. Therefore, elucidating the regulation of cell surface L1 is important for understanding the molecular mechanisms that underlie many physiological processes involved in central nervous system development. However, the molecular machinery underlying the L1 endocytic trafficking pathway that regulates the plasma membrane pool of L1 following L1-L1 homophilic binding remains unclear. Here, I report that L1 ubiquitination and dephosphorylation within the tyrosine-based motif mediate its internalization and trafficking to the lysosome in an agonist-dependent manner and that endocytosis and endocytic sorting of L1 are mediated by Rabex-5. Rabex-5 overexpression increases the rate of L1 internalization from the plasma membrane, causing an accumulation of ubiquitinated L1 on endosomes, whereas siRNAs-mediated Rabex-5 knockdown blocks L1 internalization. These results clearly indicate that L1 ubiquitination following ligand binding down-regulates cell surface L1 via the Rabex-5-mediated endocytic trafficking pathway.
Roles of Ubiquitination and the Tyrosine-based Endocytic Motif in L1 Endocytosis-The proper control of cell surface L1 expression is essential for maintaining the structural and functional homeostasis of neuronal cells. Ubiquitination is among the most widely used protein modifications involved in regulating cellular signaling and homeostasis. The detection of L1 polyubiquitination was remarkable in light of the data showing that L1 is degraded by lysosomes via the endocytic pathway, rather than by the proteasome. Recent evidence, however, suggests that multiple mono-Ub or poly-Ub moieties are associated with efficient endocytic cargo recognition (e.g. the tropomyosin-regulated kinase A receptor (TrkA), major histocompatibility complex (MHC) class I molecules, interferon (IFN)-␣/␤ receptor 1 (IFNAR1) subunit of the type I IFN receptor, and epidermal growth factor receptor (EGFR) (34 -37). The essential role of poly-Ub and multimeric Ub in the rapid internalization of transmembrane proteins suggests that polyvalent interactions are required to overcome the low affinity binding of Ub to the Ub-interacting motif of Ub-binding clathrin adaptors (38,39). Further experiments are needed to address the Ub configuration requirement for endolysosomal sorting of L1 by Rabex-5 and to identify L1-specific Ub-ligases to elucidate the function of Ub in the regulation of L1 signaling and trafficking.
Remarkably, L1 ubiquitination is required, but not sufficient, for maximal L1 internalization, which also depends on a tyrosinebased sorting motif that recruits AP-2. These data are consistent with the finding that the Src kinase inhibitor PP2 significantly enhances both L1 ubiquitination and dephosphorylation simultaneously. Thus, it is possible that the balance of Src kinase and tyrosine phosphatase activities plays a role in regulating L1 endocytosis by controlling the recruitment of a yet unidentified E3 ligase for L1. The interaction between ubiquitination and the clathrin machinery in regulating endocytosis and protein sorting is well studied in many eukaryotic cell types (40). Ubiquitinated receptors are recruited to clathrin-coated pits via interactions with the epsin and eps15 adaptor proteins (14). In addition, receptor ubiquitination may aid interaction with the AP-2 adaptor protein (36). The roles of epsin and AP-2 in regulating endocytosis of neuronal protein are well characterized (41,42). However, further experiments are required to investigate whether similar endocytic mechanisms are involved in controlling the maximal rate of L1 internalization following L1-L1 homophilic interaction.
Immunocytochemical and biotinylation studies revealed that the L1 Y1176A mutant exhibits a slower rate of internalization and a targeting to the lysosomes via the Rabex-5-mediated endocytic pathway. This raises the question of how the L1 Y1176A mutant, which does not interact with AP-2 (9), is able to internalize to the endosomes without using the clathrin-mediated endocytic pathway. Multiple internalization pathways have been associated with the uptake of ubiquitinated proteins from the cell surface. Although EGFR is exclusively internalized by the clathrin-dependent pathway in the presence of low EGF concentrations, EGFR is also internalized by clathrin-independent pathway only when exposed to higher concentrations of EGF (43). It is, therefore, possible that the L1 Y1176A mutant, which exclusively depends on ubiquitination, is internalized through a different endocytic route such as the clathrin-independent endocytic trafficking pathway.
Novel Endocytic Trafficking Pathway of Ubiquitinated L1 Is Regulated by Rabex-5-UBPs tether ubiquitinated cargo to components of the endocytic and sorting machinery, thereby enabling receptor internalization and postinternalization sorting. Although L1 is constitutively internalized into the endosomes and/or lysosomes even in the absence of L1-Ab, L1-Abinduced clustering further stimulates L1 ubiquitination concomitant with Rabex-5 recruitment to the plasma membrane to facilitate the ubiquitinated L1 internalization and sorting into lysosomes. Consistent with this, plasma membrane and early endosomal localization of L1 depend on the level of activated Rab5, as the activation of GEF for Rab5 increases the rate of L1 internalization. Taken together with data showing significant accumulation of ubiquitinated L1 in cells overexpressing Rabex-5, these data strongly support the hypothesis that the multidomain architecture of Rabex-5 allows it to perform dual roles in cargo sorting by Ub binding and ubiquitinated adaptors, and Rab5-dependent internalization. Previous studies using Vps9p, the yeast homolog of Rabex-5, indicate a possible cross-talk between the Ub-binding activity of the Coupling of Ub conjugation to ER degradation (CUE) domain and GEF activity of the VPS9 domain in the ubiquitinated cargo trafficking pathway (44,45). Based on these data, I propose the hypothesis that ubiquitinated L1 binding to Rabex-5 triggers Rab5 GEF activation through an allosteric mechanism. Further experiments are required to test this hypothesis.
Differential Roles of Two Independent UBDs in Rabex-5-To date, it remains unclear how Rabex-5 discriminates between its two UBDs, ZnF and MIU, depending on the biological context and how these UBDs cooperate with each other to transmit Ub-dependent signaling. A previous study showed that Rabex-5 binding to ubiquitinated EGF receptor in EGF-stimulated cells is dependent on both UBDs. Mutation of either domain alone had little impact on EGF receptor association, whereas mutation of both abolished the EGF receptor interaction (46). Here, I provide the first direct evidence that MIU, but not ZnF, binds and transports ubiquitinated cargo from the plasma membrane. Furthermore, the impact of the MIU domain on L1 internalization is significantly greater than the ZnF domain, despite both domains being required for membrane targeting. Unexpectedly, ZnF, which possesses Ub-ligase activity and promotes Ras ubiquitination to attenuate the Ras signaling pathway (47), does not catalyze L1 ubiquitination, indicating that Rabex-5 Ub-ligase activity may exhibit substrate specificity. In the case of L1 endocytic trafficking, Rabex-5 functions as a UBP to ensure proper internalization and intracellular sorting, rather than as an E3 Ub-ligase.
In conclusion, this study into L1 down-regulation has provided novel information into the membrane trafficking mechanism by which the Rabex-5 UBP modulates expression of a neuronal cell adhesion molecule. The proposed Rabex-5-dependent endocytosis mechanism may be applicable to a broad spectrum of cell surface receptors, including the EGF receptor.