Department of Surgery, Burn and Shock Trauma Research Institute, Loyola University Chicago Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL, 60153, USA.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S Ashland, Chicago, IL, 60607, USA.
Mol Cell Biochem. 2017 Oct;434(1-2):143-151. doi: 10.1007/s11010-017-3044-7. Epub 2017 Apr 28.
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 (EC > 10 μM), whereas the EC for CXCL12 was 4.6 nM (95% confidence interval 3.1-6.1 nM). Both agonists induced similar chemical shift changes in the C-H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the C-H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.
趋化因子（C-X-C 基序）受体 4（CXCR4）调节细胞迁移，在免疫系统中发挥重要作用。最近发现泛素是 CXCR4 的内源性非同源激动剂，它通过与 CXCR4 的配体 C-X-C 基序趋化因子配体 12（CXCL12）不同的相互作用位点激活 CXCR4。与 CXCL12 相比，泛素在原代人细胞中的趋化活性特征较差。此外，缺乏对 CXCR4 激动剂功能选择性的证据，并且泛素与 CXCR4 结合的结构后果尚不清楚。在这里，我们表明泛素和 CXCL12 在正常人外周血单核细胞、单核细胞、血管平滑肌和内皮细胞中具有相当的趋化活性。CXCR4 配体的趋化活性可以被选择性 CXCR4 拮抗剂 AMD3100 和 CXCR4 的第二跨膜结构域的肽类似物抑制。在人单核细胞中，泛素和 CXCL12 诱导的趋化作用可以被百日咳毒素和磷脂酶 C、磷脂酰肌醇 3 激酶和细胞外信号调节激酶 1/2 的抑制剂抑制。两种激动剂均可诱导血管平滑肌细胞中肌醇三磷酸的产生，该产生可被 AMD3100 抑制。在β-arrestin 募集测定中，泛素不能充分募集β-arrestin2 到 CXCR4（EC>10 μM），而 CXCL12 的 EC 为 4.6 nM（95%置信区间 3.1-6.1 nM）。两种激动剂均在膜中引起 CXCR4 的 C-H-异核单量子相关（HSQC）谱的相似化学位移变化，而 CXCL11 则未显著改变 CXCR4 的 C-H-HSQC 谱。我们的研究结果表明泛素是 CXCR4 的偏向激动剂。
