期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 46, 页码 18013-18018出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0701330104
关键词
EGFR; signal transduction; tyrosine phosphorylation
Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s(-1) and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据