Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK activation

Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, Tennessee 37208, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2009; 284(9):5742-52. DOI: 10.1074/jbc.M808507200
Source: PubMed


The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.

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    • "Interestingly, CXCL12 was shown to recruit the de-ubiquitylation enzyme USP14 to CXCR4, thereby regulating CXCR4 degradation and cell migration (Mines et al., 2009). In addition, depletion of the de-ubiquitylation enzyme USP8 was found to stabilize CXCR4 surface expression without affecting receptor ubiquitylation, indicating a role of USP8 in CXCR4 trafficking and degradation (Berlin et al., 2010). "
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    • "Additionally, deubiquitination events, primarily attributed to USP14, appear to be critical in controlling ligand-dependent CXCR4 internalization, degradation, and chemotaxis. While it was reported that over-expression of USP14 followed by CXCL12 treatment resulted in a significant reduction in CXCR4-dependent migration (Mines et al., 2009), little is known about the impact of USP14 inhibition on CXCR4 surface expression in T cells in general and specifically during aging. We now report that age-associated alteration in the expression of CXCR4 in T cells from the elderly appears to "
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    ABSTRACT: Chemokine-dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue-specific trafficking of memory-effector T cells. Previous studies carried out in rodents have demonstrated age-associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4(+) T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4(+) T cells from elderly human donors, relative to those from the young. Additionally, CXCL12-mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T-cell migration to chemotactic stimuli in the elderly.
    Aging cell 05/2012; 11(4):651-8. DOI:10.1111/j.1474-9726.2012.00830.x · 6.34 Impact Factor
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    • "CXCR4 ubiquitination occurs after receptor internalization and is mediated by the E3 ubiquitin ligase AIP4 via its interaction with β-arrestin [44], highlighting a novel function of β-arrestins in endosomal sorting of GPCRs. Deubiquitination of CXCR4, and therefore its escape from degradation, has been shown to be mediated by USP14 [45], while the deubiquitinating enzyme (DUB) USP8 has been shown to participate indirectly on CXCR4 regulation by modulating the dynamics of the signaling endosomes [46]. Similar to CXCR4, CXCR7 contains several intracellular lysines. "
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    ABSTRACT: The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.
    PLoS ONE 03/2012; 7(3):e34192. DOI:10.1371/journal.pone.0034192 · 3.23 Impact Factor
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