Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)
Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)
复制标题
血小板生成素受体胞外域中参与血小板生成素和核分布蛋白（人 NUDC）结合的残基的鉴定
DOI：
10.1074/jbc.m110.120956
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发表时间：
2010-08-20
影响因子：
4.8
通讯作者：
Xu, Peilin
中科院分区：
文献类型：
--
作者：
Chen, Wei-Min;Yu, Bo;Xu, Peilin
血小板生成素（TPO）及其受体（Mpl）与巨核细胞的增殖、分化和血小板形成有关。然而，研究还表明，Mpl的胞外结构域（Mpl-EC）与人（h）NUDC相互作用，所述NUDC是先前被表征为真菌核迁移蛋白的人同源物的蛋白质。进行该研究以进一步描绘Mpl受体上的推定结合结构域。使用酵母双杂交系统测定和免疫共沉淀，我们鉴定了在Mpl-EC结构域1（Mpl-EC-D1）内，氨基酸102-251强烈参与配体结合。我们随后用T7噬菌体展示在该区域内表达了五个亚结构域。酶联免疫吸附剂结合测定鉴定了位于残基206和251之间的短肽段作为TPO和hNUDC的最小结合结构域。随后使用该区域中的一系列连续Ala置换突变来鉴定最参与配体结合的特定残基。我们的结果指出两个疏水残基Leu（228）和Leu（230）对hNUDC结合具有实质性影响。对于TPO结合，残基Asp（235）和Leu（239）中的突变对结合效力具有最大影响。此外，保守基序WGSWS的缺失降低了hNUDC的结合能力，但不降低TPO的结合能力。TPO和hNUDC的Mpl受体上的这些单独的结合位点引起了对精氨酸-受体相互作用的有趣的影响。
Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102-251 were strongly involved in ligand binding. We subsequently expressed five sub-domains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu(228) and Leu(230), as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp(235) and Leu(239) had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.