Whitson Kristin B, Beechem Joseph M, Beth Albert H, Staros James V
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
Anal Biochem. 2004 Jan 15;324(2):227-36. doi: 10.1016/j.ab.2003.09.023.
We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.
我们制备并表征了一种新型的小鼠表皮生长因子（EGF）荧光衍生物，即Alexa Fluor 594标记的EGF（A-EGF），用于EGF-EGF受体相互作用的荧光研究。我们描述了这种衍生物的合成及其物理和生物学特性。A-EGF的激发光谱和发射光谱之间存在显著重叠，这使得该探针非常适合荧光共振能量同型转移。利用时间分辨荧光来检测EGF受体的寡聚状态，我们观察到细胞中与EGF受体结合的A-EGF存在共振能量同型转移，但膜泡中与EGF受体结合的A-EGF则没有。我们的结果结合近期EGF受体配体结合域的晶体学研究进行解释，表明观察到的荧光共振能量转移并非源于受体二聚体内的转移，而是源于高阶寡聚体内的转移。此外，我们的结果支持一种EGF受体寡聚化的结构模型，其中二聚体相对于配体结合位点头对头排列，这与通过X射线晶体学在相邻受体二聚体之间观察到的头对头相互作用一致。
