Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation

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.The CXCR4 (CXC chemokine receptor 4) is a member of the chemokine receptor family, which belongs to the superfamily of G protein-coupled receptors (GPCRs)² (1). Its ligand, CXCL12, also known as SDF-1α, also binds to RDC1, another chemokine receptor that is being proposed to be renamed as CXCR7 (2). CXCR4 mediates CXCL12-induced migration of peripheral blood lymphocytes (3), CD34⁺ progenitor cells (4), and pre- and pro-B cell lines (5). CXCR4 also plays an important role in the development of the immune system, because mouse embryos lacking either expression of the CXCR4 receptor or of its CXCL12 ligand are embryonic lethal and also manifest abnormalities in B cell lymphopoiesis and bone marrow myelopoiesis (3, 6, 7). The altered cerebellar neuron migration in mice null for the CXCR4 receptor also suggests a role for this receptor in central nervous system development. Abnormal expression and/or function of CXCR4 have been implicated in a number of diseases, including human immunodeficiency virus infection (8), cardiovascular disease (9), allergic inflammatory disease (10), neuroinflammation (11), neurodegenerative diseases (12, 13), and cancers (14-24).Stimulation of CXCR4 triggers various intracellular signaling cascades (1, 14, 25-27), such as extracellular signal-regulated kinase (ERK), which likely contribute to CXCR4-induced cell proliferation, differentiation, and/or migration. Ligand stimulation of CXCR4 also induces endocytosis of these receptors, which are targeted to lysosomes for degradation through a pathway involving ubiquitination of the C-terminal lysine residues (28). CXCR4 ubiquitination can be catalyzed by a member of the HECT family of E3 ligases, AIP4 (atrophin-interacting protein 4) (29, 30). The ubiquitinated CXCR4 is delivered to the endosomal compartments via a regulated pathway involving several adaptor proteins (31).It has been noted that deubiquitination also regulates the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes, which catalyze the removal of ubiquitin from ubiquitin-conjugated proteins, represent the largest family of enzymes in the ubiquitin system, implying the possibility that substrate selectivity is even greater for these enzymes than for those that catalyze ubiquitin ligation. Little is known about the mechanisms of CXCR4 deubiquitination and their regulation by receptor ligands. A proteomics study revealed that the steady state level of USP14 was increased upon CXCL12 stimulation of target cells (32), and preliminary studies revealed that ligand stimulation led to enhanced association of USP14 with the CXCR4. The present studies were undertaken to ascertain the functional consequences of this interaction, the selectivity of CXCR4 for USP14, when compared with three other deubiquitinating enzymes, USP2a, USP4, and USP7, and the impact of modifying the ubiquitinated state of the receptor on CXCR4 turnover, CXCL12-evoked chemotaxis, and CXCL12-induced activation of ERK.