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

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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 second 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.

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Funding

This work was supported by the National Institute of General Medical Sciences (Awards R01GM107495); by the National Cancer Institute (Award R01CA188427); by the National Heart, Lung, and Blood Institute (Award R21HL118588); and by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award No. W81XWH-15-1-0262. The content is solely the responsibility of the authors.

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Authors and Affiliations

  1. Department of Surgery, Burn and Shock Trauma Research Institute, Loyola University Chicago Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL, 60153, USA

    Jonathan M. Eby, Lauren J. Albee, Abhishek Tripathi, Xianlong Gao & Matthias Majetschak

  2. Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S Ashland, Chicago, IL, 60607, USA

    Hazem Abdelkarim & Vadim Gaponenko

  3. Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Brian F. Volkman

  4. Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL, 60153, USA

    Matthias Majetschak

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  1. Jonathan M. Eby
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Correspondence to Matthias Majetschak.

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Eby, J.M., Abdelkarim, H., Albee, L.J. et al. Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists. Mol Cell Biochem 434, 143–151 (2017). https://doi.org/10.1007/s11010-017-3044-7

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