Modulation of the CXC Chemokine Receptor 4 Agonist Activity of Ubiquitin through C-Terminal Protein Modification

Abstract

Extracellular ubiquitin has recently been described as a CXC chemokine receptor (CXCR) 4 agonist. Studies on the structure–function relationship suggested that the C-terminus of ubiquitin facilitates CXCR4 activation. It remains unknown, however, whether C-terminal processing of ubiquitin could be biologically relevant and whether modifications of the ubiquitin C-terminus can modulate CXCR4 activation. We show that C-terminal truncated ubiquitin antagonizes ubiquitin and stromal cell-derived factor (SDF)-1α induced effects on cell signaling and function. Reduction of cell surface expression of insulin degrading enzyme (IDE), which cleaves the C-terminal di-Gly of ubiquitin, enhances ubiquitin induced reduction of cAMP levels in BV2 and THP-1 cells, but does not influence changes in cAMP levels in response to SDF-1α. Reduction of cell surface IDE expression in THP-1 cells also increases the chemotactic activity of ubiquitin. As compared with native ubiquitin, C-terminal Tyr extension of ubiquitin results in reduced CXCR4 mediated effects on cellular cAMP levels and abolishes chemotactic activity. Replacement of C-terminal di-Gly of ubiquitin with di-Val or di-Arg enhances CXCR4 mediated effects on cAMP levels and the di-Arg substitution exerts increased chemotactic activity, when compared with wild type ubiquitin. The chemotactic activities of the di-Val and di-Arg mutants and their effects on cAMP levels can be antagonized with C-terminal truncated ubiquitin. These data suggest that the development of CXCR4 ligands with enhanced agonist activities is possible and that C-terminal processing of ubiquitin could constitute a biological mechanism, which regulates termination of receptor signaling.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

“…Intracellular Ca 2+ in A7r5 cells was measured using the Fluo-4 NW Calcium Assay Kit (Molecular Probes), as described (51,52,79). Intracellular Ca 2+ in human VSMC was measured using the ratiometric Ca 2+ indicator dye Fura-2.…”

“…FACS analyses of receptor cell-surface expression were performed as described previously (51,79). Cells were labeled with polyclonal rabbit anti-CXCR4 (ab2074, Abcam), polyclonal rabbit anti-α 1A AR (ab137123, Abcam), and polyclonal rabbit anti-α 1B AR (ab169523, Abcam) in combination with anti-rabbit FITC-conjugated goat IgG (ab6717, Abcam).…”

“…Western blotting with mouse monoclonal anti-phospho-MLC2 (S19) (Cell Signaling), rabbit polyclonal anti-CXCR4 (ab2074, Abcam), rabbit polyclonal anti-α 1A AR (ab137123, Abcam), rabbit polyclonal anti-α 1B AR (ab169523, Abcam), and mouse anti-α 2C AR (ab167433, Abcam) in combination with anti-rabbit or mouse IgG horseradish peroxidase-linked whole antibody (GE Healthcare) was performed as described (52,79). Rabbit anti-GAPDH (Cell Signaling) in combination with anti-rabbit IgG horseradish peroxidase-linked whole antibody (GE Healthcare) was used as a protein loading control.…”

The Intricate Role of CXCR4 in Cancer

“…The chemokine stromal cell-derived factor-1, now renamed as CXCL12, was established as the specific ligand for CXCR4 (Bleul, Fuhlbrigge, Casasnovas, Aiuti, & Springer, 1996; Oberlin et al, 1996). Although CXCL12 is the only known chemokine that binds CXCR4, recent studies suggest that extracellular ubiquitin also acts as an immune modulator through CXCR4-mediated signaling (Saini, Marchese, & Majetschak, 2010; Tripathi et al, 2013). Although CXCR4 is known to bind only CXCL12, in 2005 another chemokine receptor CXC receptor 7 (CXCR7, ACKR3, RDC1, CMKOR1, or GPR159) was established as a receptor for CXCL12 (Balabanian et al, 2005; Burns et al, 2006).…”

CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin

“…Recently, we and others have reported that extracellular ubiquitin functions as an immune modulator and as another CXCR4 agonist [1–4]. Unlike SDF-1α, however, ubiquitin does not bind to CXCR7 and both ligands appear to activate CXCR4 through different mechanisms and separate binding sites on the receptor [5–7]. Both CXCR4 ligands are constitutively expressed in the systemic circulation and increased concentrations of SDF-1α and ubiquitin have been reported during infectious and sterile inflammation [1].…”