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Access personal reporting. More About Us. 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 Atype 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 L1 WT and L1 YA , a tyrosine-based motif mutant, but not L1 K11R , an ubiquitination-deficient mutant, leading to the accumulation of ubiquitinated L1 on endosomes. Overall, these results provide a novel mechanistic insight into how Rabex-5 regulates internalization and postendocytic trafficking of ubiquitinated L1 destined for lysosomal degradation.
Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases
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 , — , 5. The endosomal trafficking is also required to target L1 properly to the growing axon.
L1 endocytosis is substrate-dependent process and occurs as a consequence of L1-L1 homophilic binding. 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 , — , 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 , — , A number of UBPs, including Rabex-5, present at endosomes are able to participate in degradative sorting of ubiquitinated cargo 14 , — , Rabex-5, originally identified as a rabaptininteracting protein, possesses GEF activity for Rab5 It is a multidomain protein consisting of an Atype zinc finger ZnF fused to a motif interacting with Ub MIU domain at the N terminus, membrane-binding motif, and tandem helical bundle-VPS9 domains at the center, and a coiled coil region at the C terminus.
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 , 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 Interfering with L1 ubiquitination reduces L1 internalization due to impairment of the interaction with Rabex It is, therefore, plausible that Rabex-5 plays an important role in neuronal function by controlling L1 internalization and endocytic sorting.
Affinity-purified polyclonal anti-Rabex-5, anti-Rab5, and anti-L1 antibodies were raised using a C-terminal fragment of bovine Rabex-5 residues — , full-length human Rab5a, and full-length rat L1 as immunogens, respectively. Mouse monoclonal anti-L1 antibody H7 , rat monoclonal anti-mouse L1 antibody, and rabbit polyclonal anti-human L1 antibody were provided by Dr. Secondary antibodies conjugated to HRP were purchased from Pierce.
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Bafilomycin A1, MG, and lactacystin synthetic were obtained from Calbiochem. All constructs were confirmed by DNA sequencing. Scrambled siRNAs for Rabex-5 were used as negative controls. Soluble extracts were incubated with goat anti-L1, anti-Myc, or anti-FLAG antibodies for 5 h, and then protein G-Sepharose beads were added and incubated for a further 1 h. Immunoprecipitated complexes were washed six times with lysis buffer, and bound proteins were eluted with SDS sample buffer.
After extensive washing, immunoreactivity was detected using an enhanced chemiluminescence detection kit Pierce. The pellet was resuspended in fractionation buffer.
Excess biotin was quenched by washing with DMEM. Remaining cell surface biotin was stripped using stripping solution 50 m m glutathione, 0. After washing five times with cell lysis buffer, the bound proteins were removed with SDS sample buffer. To examine colocalization of fluorescence signals in different channels, the MetaMorph colocalization function following background subtraction and threshold setting were used.
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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. Error bars denote the S. Previous studies on L1 demonstrated that cell adhesion can be spatially regulated by the polarized internalization and recycling of cell adhesion molecules 3 , 4.
Indeed, time lapse imaging directly showed that internalization of L1-GFP localized on the plasma membrane into intracellular vesicles depends on cell adhesion in N2a cells Fig.
Crosstalk and Integration of Membrane Trafficking Pathways
To gain insight into the molecular machinery underlying cell adhesion-dependent internalization and postendocytic sorting of L1, I investigated L1 endocytosis induced by incubation with L1-Ab, which mimics L1-L1 homophilic binding 9 , 24 in N2a cells. Immunocytochemical analysis showed that endogenous L1 was localized predominantly to the plasma membrane in untreated cells The percentage of intracellular to total fluorescence intensity of L1 increased with an increase in the L1-Ab incubation time, i.
Image quantification confirmed that 4. L1 ligand-dependent internalization of L1 in N2a cells. A , time-lapse imaging showing cell adhesion-dependent internalization of L1-GFP arrows in N2a cells upper panels. Note that L1-GFP accumulates on the lamellipodium tip before the cells adhere to each other arrowheads. White boxed areas are enlarged middle panels. Pixel intensity was determined by a row average plot of a section selected from a confocal slice through the center of the cell. PM , plasma membrane.
B , L1-Ab-induced endocytosis of L1 in N2a cells. N2a cells were labeled with Alexa Fluor conjugated L1 antibodies green. Arrows indicate internalized L1. Ubiquitination, lysosomal targeting, and degradation of L1 after incubation with L1-Ab. A , superimposed images show the colocalization of internalized L1 red and GFP-Rab5 green -positive early endosomes or GFP-Rab 11 green -positive recycling endosomes upper panels. Arrows indicate the L1 colocalization with Rab5- or Rabpositive vesicles. Lysosomes are shown in red , and colocalized components are shown yellow arrows.
B , N2a cells were incubated in the presence of L1-Ab. After cell lysis, immunoprecipitation IP and immunoblot analyses were performed as indicated.
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The blots were stripped and reprobed with anti-FK1 antibody that recognizes polyubiquitination right panel. To control the functionality of the antibodies cell lysates were applied Lysate.
http://aktiv-vita.ru/modules After cell lysis, immunoprecipitation and immunoblot analyses were performed. Dephosphorylation dY of tyrosine Y in the YRSL motif within the cytoplasmic domain of L1 was detected using the H7 monoclonal antibody 9 left panel.
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Arrows and arrowheads indicate internalized L1 and accumulated L1 on plasma membrane, respectively middle panels. N2a cells coexpressing the indicated plasmids were incubated in the presence or absence of L1-Ab. After cell lysis, immunoprecipitation and immunoblot analyses were performed lower. Equal amounts of cell lysates were immunoblotted with L1-Ab upper. Degradation kinetics of L1 were determined by densitometry of immunoblots illustrated in the upper panels lower. Consistent with previous results 9 , 25 , L1-Ab treatment led to L1 monoubiquitination Fig.
Furthermore, weak but detectable bands were observed upon L1-Ab treatment with FK1 antibody that recognizes polyubiquitination Fig. 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.
Surprisingly, the extent of L1 ubiquitination in PP2-pretreated cells significantly increased by 1. 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 Furthermore, we also map the transcription factors, gene ontology, and gene specific enriched reactome and KEGG pathways information on the reconstructed direct sub-network to infer direct physical and genetic interactions between perturbed genes and their effected components Fig.
We primarily focus on Ras and PI3K-Akt signaling pathways, to study and infer cross-talks among genetic components between them. We carried out Gene Ontology and pathway enrichment analysis of the complete list of perturbed genes, for which corresponding expression profiles are utilized in this study.
It includes unique perturbed genes which have regulatory effects on the other genes. The functional term enrichment analysis suggests the role of these genes in intracellular protein transport such as exocytosis and endocytosis.
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