CXCR4: A Receptor for Extracellular Ubiquitin

Ubiquitin (Ub) is a small, regulatory protein that is highly conserved in all eukaryotes.^{1, 2} Covalent attachment of Ub to intracellular proteins is referred to as ubiquitination. Ubiquitination is a multistep enzymatic process that requires E1 activating, E2 conjugating, and E3 ligating enzymes, while removal of Ub is carried out by a family of deubiquitinating enzymes (DUBs).^{3, 4} Modulating the activity of any of these enzymes can potentially regulate Ub-related pathways and activities.

Ubiquitination typically marks proteins for proteasomal degradation, but it can also be involved in regulating processes such as protein localization, enzyme activity, and protein-protein interactions.^{3, 4} Whether a protein is monoubiquitinated or polyubiquitinated, and the types of Ub linkages that form the poly-Ub chains, can also have varying effects on protein function.^{4, 5} The role of ubiquitin is further complicated by increasing evidence suggesting that it may also have an extracellular function. Extracellular Ub can regulate cell growth and apoptosis, induce antimicrobial activities, and modulate immune responses.^{6, 7, 8, 9, 10, 11, 12, 13, 14, 15} In addition, Ub is known to inhibit the production of the pro-inflammatory cytokine TNF-alpha and enhance the production of IL-4, IL-10, and IL-13.^{10, 11, 12, 13, 14, 15} However, the mechanism underlying these effects has remained unclear.

Recent studies by Saini and colleagues provided evidence to suggest that Ub has the ability to activate a membrane receptor.^{16, 17, 18} Fluorescently labeled Ub exhibits typical receptor binding characteristics with a Kd of approximately 100 nM.^{16, 17} In addition, exogenous Ub can stimulate cellular responses characteristic of receptor activation, including Ca²⁺ fluxes, decreases in intracellular cAMP, and downstream phosphorylation of RSK1, Akt, and ERK1/2.^{16, 17, 18} Ub-induced Ca²⁺ signals are blocked by both pertussis toxin (a G alpha_i/o inhibitor) and U73122 (a phospholipase C inhibitor), suggesting that Ub activates G protein-coupled receptor (GPCR) signaling pathways.^{16, 17} Using a combination of bioassays, pharmacology, siRNA knockdown, and receptor competition experiments, the putative Ub receptor was identified as CXCR4.^{16, 17}

CXCR4 is a GPCR best known for mediating the activities of the chemokine CXCL12/SDF-1, a potent chemotactic agent for leukocytes.¹⁹ CXCR4 can also bind to macrophage migration inhibitory factor (MIF), act as a CD4 co-receptor for HIV-1, and contribute to in cancer progression via its effects on cell motility, cell survival, and angiogenesis.^{19, 20, 21} In many respects, the in vitro biological activities of CXCL12 and Ub appear to be similar. Both stimulate the activation of G alpha_i/o, promote Ca²⁺ mobilization, and induce a similar pattern of kinase phosphorylation.^{16, 17, 18, 20} Both proteins have also been shown to induce chemotaxis in vitro, although Ub appears to be somewhat less potent.^{18, 20} In addition, kinase signaling and the effects on cell motility induced by Ub and CXCL12 are blocked by the CXCR4 inhibitor AMD3100.¹⁸ Despite these similarities, CXCL12 and Ub exert their activities through distinct interactions with CXCR4. CXCL12-induced signaling requires an initial interaction with the N-terminal region of CXCR4, followed by binding to extracellular loops 2 and 3.^{18, 19} Ub appears to associate with the same extracellular loops, but does not require binding to the N-terminus.¹⁸ In addition, unlike CXCL12, Ub does not interfere with HIV-1 binding to CXCR4 and does not have the ability to suppress infectivity.¹⁸

The interest in understanding the activities of Ub is expanding rapidly. Despite being initially defined as an extracellular factor, the majority of research has focused on its intracellular roles.² Studies identifying CXCR4 as a physiological receptor for Ub may reveal new and exciting avenues of research, including establishing potential mechanisms for the effects that extracellular Ub appears to have on immune system function.^{9, 10, 11, 12, 13, 14, 15}

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Extracellular Ubiquitin is a Noncognate Ligand for the Chemokine Receptor CXCR4. Research has suggested that extracellular Ubiquitin (Ub) modulates immune responses. Extracellular Ub levels are elevated during inflammation as the result of its release from damaged tissues and lysed erythrocytes, as well as its active secretion from chromaffin cells of the adrenal gland during the stress response. Ub binds to and signals through CXCR4, a G alphai/o-protein coupled receptor that signals through multiple pathways including cAMP/PKA, PLC-beta, and PI 3-Kinase/Akt. CXCR4 activation could possibly mediate the anti-inflammatory effects induced by extracellular Ub. Following receptor binding, Ub is internalized via receptor-mediated endocytosis and used to replenish the pools of free Ub for the Ub-proteasome pathway. Thus, extracellular Ub could potentially produce a secondary cell signal by modifying intracellular proteins.

References

1. Ozkaynak, E. et al. (1984) Nature 312:663.
2. Goldstein, G. et al. (1975) Proc. Nat. Acad. Sci. USA 72:11.
3. Ben-Neriah, Y. (2002) Nat. Immunol. 3:20.
4. Schnell, J.D. & L. Hicke (2003) J. Biol. Chem. 278:35857.
5. Behrends, C. & J.W. Harper (2011) Nat. Struct. Mol. Biol. 18:520.
6. Daino, H. et al. (2000) Blood 95:2577.
7. Singh, M. et al. (2010) Cardiovasc. Res. 86:20.
8. Kieffer, A.E. et al. (2003) FASEB J. 17:776.
9. Nakamura, M. et al. (1996) J. Immunol. 156:533.
10. Majetschak, M. et al. (2003) Blood 101:1882.
11. Majetschak, M. et al. (2004) Surgery 135:536.
12. Majetschak, M. et al. (2004) J. Trauma 56:991.
13. Patel, M.B. et al. (2006) J. Surg. Res. 135:226.
14. Garcia-Covarrubias, L. et al. (2008) Crit. Care Med. 36:979.
15. Majetschak, M. (2011) J. Leukoc. Biol. 89:205.
16. Saini, V. et al. (2010) J. Biol. Chem. 285:15566.
17. Saini, V. et al. (2010) Commun. Integr. Biol. 3:608.
18. Saini, V. et al. (2010) J. Biol. Chem. 286:33466.
19. Busillo, J.M. & J.L. Benovic (2007) Biochim. Biophys. Acta. 1768:952.
20. Teicher, B.A. & S.P. Fricker (2010) Clin. Cancer Res. 16:2927.
21. Bernhagen, J. et al. (2007) Nat. Med. 13:587.
22. Majetschak, M. et al. (2006) Immunol. Cell Biol. 84:59.

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