It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

中文翻译：

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全长人类Grb7蛋白和磷酸化代表突变体的表征。

众所周知，受体酪氨酸激酶（RTK）的二聚化状态与诸如生长因子受体结合蛋白7（Grb7）蛋白之类的结合伴侣一起在细胞信号调节中起着重要作用。先前，我们提出在RTK的下游，Grb7SH2域酪氨酸残基的磷酸化状态可以控制Grb7二聚化，而二聚化可能是Grb7与RTK结合的重要调控步骤。以这种方式，在RTK介导的信号通路中，膜结合激酶的下游可能会发生其他二聚化依赖性调节。大量纯净和稳定的FL蛋白的存在阻碍了全长（FL）Grb7蛋白的外推以及进一步检验该假设的能力。这里，我们报告了FL Grb7蛋白的生物物理特征，也代表了酪氨酸磷酸化的Grb7蛋白形式的突变体。通过尺寸排阻色谱和分析超速离心，我们显示了磷酸化酪氨酸模拟Y492E-FL-Grb7蛋白（Y492E-FL-Grb7）在预期的生理浓度下基本上是单体的。先前已证明野生型FL Grb7（WT-FLGrb7）蛋白是二聚体，其解离常数（Kd）约为11μM。我们的研究在这里测量了WT-FL-Grb7的FL蛋白二聚化Kd，其数量级约为1μM。通过动态光散射法确定了WT-FL-Grb7与酪氨酸磷酸化模拟物Y492E-FL-Grb7蛋白相比的近似大小和形状。Grb7SH2结构域的体外磷酸化表明，只有一个可用的酪氨酸残基被磷酸化，表明相同的磷酸化模式可能与FL蛋白有关。总体上解释了生物物理表征研究，以期了解功能活跃的Grb7蛋白构象。