Departments of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA.
Biochemistry. 2012 Jul 17;51(28):5642-54. doi: 10.1021/bi3001566. Epub 2012 Jul 2.
MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 Å resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.
MIP-2/CXCL2 是一种与人类趋化因子相关的鼠类趋化因子，具有 Glu-Leu-Arg（ELR）激活基序，并激活 CXCR2 以引起中性粒细胞趋化。我们解析了 MIP-2 的结构，分辨率为 1.9Å，并基于人源 CXCR4 的坐标构建了其鼠源受体 CXCR2 的模型。细胞通过特定的 G 蛋白偶联受体趋化因子诱导迁移受到糖胺聚糖（GAG）的调节，GAG 会使趋化因子寡聚化。通过 NMR 光谱鉴定了 MIP-2 与肝素二糖 I-S 相互作用的 GAG 结合残基。创建了支持趋化因子与受体之间 2:2 复合物的模型 GAG/MIP-2/CXCR2 复合物。这些二糖结合残基的突变体被构建并测试了肝素结合、体外中性粒细胞趋化性以及体内中性粒细胞募集到小鼠腹膜和肺部的能力。突变体的体外中性粒细胞趋化性降低了 10 倍。在腹膜中，野生型 MIP-2 和突变体之间的中性粒细胞募集没有差异，但突变体在肺部的所有活性都丧失了，这支持了 GAG 对趋化因子的调节是组织依赖性的概念。
