Molecular mechanisms for plasma membrane localization of human transmembrane receptors
D'Agostino, Massimo (2011) Molecular mechanisms for plasma membrane localization of human transmembrane receptors. [Tesi di dottorato] (Inedito)
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I° Abstract: E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 a-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8a in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8a. Moreover, CD8a redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8a ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8a localization at the plasma membrane by ubiquitylation and endocytosis. II° Abstract: Familial exudative vitreorethinopaty (FEVR) is an hereditary ocular disorder caused by an insufficient vascularization of the periferal retina during development. The resulting hypoxia actives a compensatory vascularization but the new vessels are prone to rapture causing exudates and bleeding followed by scarring, retinal detachment and blindness. Mutations in Frizzled4 (Fz4), a member of the cell surface Wnt family receptors, were found in many FEVR patients. In a autosomal dominant form of FEVR (the most frequent and henceforth referred as Fz4-FEVR), the deletion of two nucleotides leads to the synthesis of a completely different and truncated cytosolic tail (L501fsX533). This receptor is retained in the ER and somehow traps Fz4 by oligomerization, thus performing its dominant effect. We confirmed the ER retention of Fz4-FEVR in transfected cells and observed the formation of oligomers of high molecular weigth in a time dependent manner, suggesting that oligomerization plays a role in the localization of the mutant. Interestingly, when we replaced the cytosolic tail of the G glycoprotein coded by the ts-045 mutant strain of VSV (VSVG) with the tail of wilde-type or mutant receptor, the chimeric VSVG-Fz4-FEVR was completely retained in the ER, whereas VSVG-Fz4 exited from the ER. Thus, the Fz4-FEVR tail is sufficient to determine ER localization. In addition, performing a competition experiment, we found that overexpression of a peptide coding FEVR tail resulted in the recovery of the cell surface expression of Fz-FEVR, suggesting that its retention in the ER is protein-mediated. Most importantly, we found with a proteomic approach a new interactor of both Fz4wt and Fz4-FEVR, the αB-Crystallin protein. This interaction was not tail dependent, suggesting that the three cytosolic loops of the receptor may be involved. αB-Crystallin overexpression promoted cell surface expression of Fz4-FEVR and prevented its dominant negative effect on the wild-type counterpart. Thus, αB-Crystallin might represent an important tool to contrast FEVR. Current effort is focused to identify the specific interactor(s) responsible of the retention in the ER of Fz4-FEVR in order to identify others possible therapeutic target(s).
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