Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, United States.
Biochemistry. 2012 Mar 27;51(12):2551-7. doi: 10.1021/bi300200p. Epub 2012 Mar 15.
Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.
最近的研究结果表明，凝血因子 Xa（FXa）裂解蛋白酶激活受体 2（PAR-2），在内皮细胞中引发保护性的细胞内信号反应。在这项研究中，我们通过监测 FXa 在瞬时转染 PAR-2 裂解报告构建体的内皮细胞中对 PAR-2 的裂解，研究了 FXa 与 PAR-2 相互作用特异性的分子决定因素，其中受体的细胞外结构域融合到编码碱性磷酸酶的 cDNA 中。比较一系列 FXa 突变体裂解 PAR-2 的效率表明，39 环的酸性残基（Glu-36、Glu-37 和 Glu-39）和 60 环的碱性残基（Lys-62 和 Arg-63）、148 环的碱性残基（Arg-143、Arg-150 和 Arg-154）以及 162 螺旋的碱性残基（Arg-165 和 Lys-169）有助于 FXa 在内皮细胞上识别受体的特异性。这一点可以从突变体对转染细胞表达的 PAR-2 的活性显著降低得到证明。FXa 突变体 PAR-2 裂解活性的丧失程度与它们的 PAR-2 依赖性细胞内信号活性的丧失程度相关。对 FXa 突变体的进一步表征表明，除了在凝血酶原酶复合物的识别特异性中起作用的 162 螺旋的碱性残基外，研究中的任何表面环残基都不会显著促进 FXa 在凝血酶原酶复合物中的活性。这些结果为 FXa 如何特异性地与凝血和信号通路中的其大分子底物相互作用提供了新的见解。
