Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

Abstract

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking and structural consequences of ubiquitin binding to CXCR4 are unknown. Here we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the 2nd transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In β-arrestin recruitment assays, ubiquitin did not sufficiently recruit β-arrestin2 to CXCR4 (EC50>10 μM), whereas the EC50 for CXCL12 was 4.6 nM (95% confidence interval: 3.1–6.1 nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.

Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

“…We first tested a total of 14 AR antagonists at a concentration of 100 μM in PRESTO-Tangoβ-arrestin recruitment assays for CXCR4 and ACKR3 ( Fig 1A and 1B ). CXCL12, the natural agonist of both receptors, was used as a positive control and employed at a saturating concentration (200 nM), which is more than 40- times the EC 50 concentration for CXCL12 in this assay system [ 10 , 13 , 17 ]. Consistent with our previous findings, the luminescence signal increased 1.8- fold upon activation of CXCR4 with CXCL12 (p<0.05 vs. unstimulated cells, Fig 1A )[ 10 , 13 , 17 ].…”

“…CXCL12, the natural agonist of both receptors, was used as a positive control and employed at a saturating concentration (200 nM), which is more than 40- times the EC 50 concentration for CXCL12 in this assay system [ 10 , 13 , 17 ]. Consistent with our previous findings, the luminescence signal increased 1.8- fold upon activation of CXCR4 with CXCL12 (p<0.05 vs. unstimulated cells, Fig 1A )[ 10 , 13 , 17 ]. The CXCR4 PRESTO-Tango luminescence signals after stimulation with prazosin and the prazosin-related α 1 -AR antagonist cyclazosin increased 3.0- fold and 2.15- fold , respectively (p<0.05 vs unstimulated cells and p>0.05 vs. CXCL12 for both), suggesting that both drugs induce β-arrestin recruitment to CXCR4 with an efficacy comparable to CXCL12.…”

“…We employed nuclear magnetic resonance (NMR) spectroscopy and utilized 13 C-labeled methylated membranes prepared from cells overexpressing CXCR4 or ACKR3 to closely mimic native conditions for receptor folding and interactions with the plasma membrane. We have utilized this strategy previously to assess ligand binding to CXCR4 and α 1a -AR [ 9 , 13 ]. We selected atipamezole as a control drug that did not activate CXCR4 or ACKR3 in PRESTO-Tango assays.…”

“…Next, we addressed whether prazosin and cyclazosin also modulate CXCR4- and ACKR3-mediated hVSMC function. Because VSMCs are known to migrate towards CXCR4 and ACKR3 agonists[ 10 , 13 , 23 ], we utilized chemotactic responses of hVSMCs as a functional read-out. Neither of the drugs, however, induced chemotaxis of hVSMCs ( Fig 8A and 8B , open squares).…”

“…The assay was performed as recently described [ 7 , 11 – 13 ]. HTLA cells (2.5x10 5 /well) were seeded in a 6-well plate and transfected with 750 ng of each of the TANGO plasmids using Lipofectamine 3000 (ThermoScientific).…”