CXC Chemokine Receptor 4 Is a Cell Surface Receptor for Extracellular Ubiquitin

Abstract

Ubiquitin is one of the most highly conserved proteins in eukaryotes and plays major biological roles as a post-translational protein modifier. Ubiquitin is also a natural constituent of plasma, and several lines of evidence suggest that extracellular ubiquitin is an immune modulator with anti-inflammatory properties. In addition, ubiquitin treatment has been shown to limit inflammation and reduce organ injury in various disease models and species in vivo. However, its mechanism of action is unknown. Here we show that extracellular ubiquitin is a natural CXC chemokine receptor 4 (CXCR4 and CD184) agonist. Extracellular ubiquitin promotes intracellular Ca 2؉ flux and reduces cAMP levels through a G protein-coupled receptor that signals via a G␣ i/o protein in THP1 cells. Toll-like receptor 4 stimulation reduces ubiquitin-binding sites, which enabled identification of four G␣ i/o PCRs as ubiquitin receptor candidates. Overexpression of candidate genes in HEK293 cells, gene silencing in THP1 cells, competition binding, and signaling studies with the CXCR4 agonist stromal cell-derived factor-1␣ (chemokine (CXC motif) ligand 12) and inhibitor AMD3100 identify CXCR4 as a functional ubiquitin receptor. Our finding uncovers a fundamentally new aspect of the role of ubiquitin in biology, has implications for the understanding of CXCR4-mediated events, and is expected to facilitate development of new therapeutic avenues for a variety of diseases.

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors

“…Then, we investigated the inhibitory activity of CSB6B on the ubiquitin-protein interaction by examining the binding affinity of ubiquitin to CXCR4, a cell surface receptor that recognizes ubiquitin as a ligand [13][14][15]. Since ubiquitin binding to the cell surface would be mediated by CXCR4, the interaction of FITC-labeled ubiquitin with CXCR4 was assessed by measuring the fluorescence associated with human monocyte cells (THP-1) after treating CSB6B at various concentrations (Figs.…”

“…Thus, we tested the effect of CSB6B in the extracellular space. It has been explored that the extracellular ubiquitin binds to CXCR4 as a ligand and turns on CXCR4 signaling, which results in enhanced AKT expression, AKT phosphorylation, and metastasis [13,31]. In the ubiquitin-CXCR4 signaling process, it is known that hydrophobic residues of ubiquitin including Phe4 and Val70 directly bind CXCR4, and the C-terminal motif facilitates receptor activation [31].…”

“…Recent progress in ubiquitin biology revealed that various forms of unanchored ubiquitin are also involved in cellular signaling [10][11][12]. For example, unanchored extracellular ubiquitin is responsible for apoptosis, cell growth, immune modulation, and cancer progression through its binding to CXC chemokine receptor type 4 (CXCR4) [13][14][15].…”

ISG15 regulates IFN-γ immunity in human mycobacterial disease

“…The cytokine properties of ISG15 are reminiscent of potential cytokine properties of ubiquitin and other ubiquitin-like proteins. Several studies have suggested that extracellular ubiquitins serve as immune modulator during infectious and noninfectious inflammation [14,15]. However, not many reports are available regarding other ubiquitin-like proteins.…”

SDF-1–CXCR4 differentially regulates autoimmune diabetogenic T cell adhesion through ROBO1–SLIT2 interactions in mice

“…The receptors for SDF-1 are C-X-C receptor (CXCR)7 [1] and CXCR4, the latter of which only binds SDF-1 and ubiquitin [2]. CXCR7 possesses a tenfold higher affinity for SDF-1 than CXCR4 [1]; however, it is expressed at very low levels on T cells [3].…”