The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

Abstract

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1␣ do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1␣ lead to similar G␣ iresponses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-l␣ in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1␣, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

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

“…This implies that CXCR4 and also ACKR3 function as modulators of α 1 -AR. Whereas the molecular mechanisms through which ACKR3 influences α 1 -AR function remain to be determined, the observed effects of ubiquitin in the present study are in agreement with the findings that ubiquitin functions as a noncognate CXCR4 agonist that does not bind to ACKR3 (51,52,(59)(60)(61)(62)(63). The observation that CXCL12, which has a much higher affinity for ACKR3 than for CXCR4 (64), also enhanced the potency of PE in normal animals in vivo in the present study, whereas CXCL12 previously desensitized PE-mediated vasoconstriction of isolated arteries and reduced blood pressure during hemorrhagic shock (9), suggests that effects of CXCL12 depend on the relative functional contribution of CXCR4 and ACKR3 within the specific experimental or (patho)physiological environment (65).…”

“…5A shows a typical Western blot with anti-CXCR4 with cell homogenates from human VSMC transfected with nontargeting (NT) or CXCR4-targeted siRNA. As expected, anti-CXCR4 recognized multiple bands in human VSMC transfected with NT siRNA, which likely correspond to proteolytically processed, ubiquitylated, or glycosylated forms of CXCR4 (51)(52)(53)(54)(55). The most abundant receptor species after transfection of human VSMC with NT siRNA were detectable at migration positions corresponding to 48-60 kDa.…”

“…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.…”

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.…”

“…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].…”