Effect of ErbB2 Coexpression on the Kinetic Interactions of Epidermal Growth Factor with Its Receptor in Intact Cells
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- 作者：John C. Wilkinson and James V. Staros
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 8, 2002
- 年：2002
- 卷：41
- 期：1
- 页码：8 - 14
- 全文大小：123K
- 年卷期：v.41,no.1(January 8, 2002)
- ISSN：1520-4995
文摘
We have extended the use of stopped-flow mixing and fluorescence anisotropy detection toinvestigate in real-time the effects of ErbB2 coexpression on the kinetic interactions of epidermal growthfactor (EGF) with the EGF receptor. Using stable 32D-derived cell lines expressing both the EGF receptorand ErbB2, and fluorescein-labeled H22Y murine EGF (F-EGF), a series of association and dissociationexperiments were performed in which the kinetic interaction of F-EGF with cells was monitored byobserving time-dependent changes in fluorescence anisotropy following rapid mixing. Data were collectedat various concentrations of F-EGF and multiple cell densities, using cells that express similar levels ofthe EGF receptor but different levels of ErbB2, and then analyzed by fitting to a two independent receptor-class model using global analysis techniques. The recovered kinetic parameters indicated that thecoexpression of ErbB2 had relatively modest effects on recovered rate constants and calculated Kd values,but a significant effect on the fraction of receptors associated with the high-affinity receptor class. Thiseffect on the fraction of high-affinity receptors depended on the relative expression of ErbB2, as higherErbB2 expression levels correlated with a larger fraction of high-affinity receptors. Further, the increasein the fraction of high-affinity receptors due to the presence of ErbB2 occurred without any change in thetotal number of EGF binding sites per cell. Thus, we have identified modulation of the relative populationsof high- and low-affinity classes of EGF receptors as a consequence of coexpression of ErbB2 with theEGF receptor.