Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach-猫眼课题宝

SH2 结构域与酪氨酸磷酸化血小板衍生生长因子 β 受体序列之间的相互作用：通过基于新型生物传感器的方法分析动力学参数

DOI：

10.1128/mcb.13.6.3567-3576.1993

发表时间：

1993

期刊：

Molecular and Cellular Biology

影响因子：

5.3

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

G. Panayotou;G. Gish;Peter End;Oanh Truong;Ivan Gout;R. Dhand;M. J. Fry;Ian Hiles;T. Pawson;Michael D. Waterfield

通过动力学参数的实时测量来检测SH2结构域与含磷酸酪氨酸序列之间的相互作用。磷脂酰肌醇3-激酶p85亚基的SH2结构域以及其他信号分子在细菌中以谷胱甘肽s -转移酶融合蛋白的形式表达。合成了与人血小板衍生生长因子β受体上负责磷脂酰肌醇3-激酶结合的两个自磷酸化位点相对应的含磷酸酪氨酸肽，并将其用作捕获分子，固定在生物传感器表面。测定了完整p85和重组SH2结构域结合这两个位点的结合力和解离速率常数。研究发现，在所有相互作用中，高缔合率伴随着非常快的解离率。p85的两个SH2结构域具有结合特异性，其中n端SH2与Tyr-751位点结合亲和力高，而与Tyr-740位点结合亲和力不高，c端SH2与这两个位点都有强烈的相互作用。这种方法应该普遍适用于研究活化蛋白-酪氨酸激酶受体组装信号复合物的特异性。

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.