Rotational dynamics of the epidermal growth factor receptor
Rotational dynamics of the epidermal growth factor receptor
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DOI：
10.1021/bi015733q
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发表时间：
2002-02-12
期刊：
影响因子：
2.9
通讯作者：
Beth, AH
中科院分区：
文献类型：
--
作者：
Stein, RA;Hustedt, EJ;Beth, AH
我们已经研究了SL-EGF的旋转迁移率，SL-EGF是双（磺基-N-琥珀酰亚胺基）-[N-15，H-2（16）]-doxyl-2-spiro-4 '-庚二酸酯和[Lys 3，Tyr 22]-鼠表皮生长因子的双官能加合物，结合到A431膜囊泡中的EGF受体。线性EPR谱表明，在结合的复合物制剂中基本上没有游离的SL-EGF。为了更好地定义结合到EGF受体的SL-EGF的旋转迁移率，在多个塞曼场调制频率下获得ST-EPR光谱。全球分析与单轴旋转扩散模型的ST-EPR数据产生了两个最小值，有不同的旋转流动性和方向的SL-EGF相对于膜法向轴。两个旋转物种的旋转迁移率是一致的单体和二聚体或稍大的低聚物，如三聚体或四聚体，反对高阶受体聚集在受体活化中的作用。考虑到两个最小值和先前的观察，A431膜囊泡包含两个可区分的配体结合群体，ST-EPR光谱与具有两个单轴旋转物种的模型拟合。这产生了两个组件，这是类似于从两个原始的一个组件的配合，无论是快速或缓慢的旋转流动性，不同的方向。模型依赖的结果表明，有潜在的构象和旋转的差异，在这两个人口，并提供了一个合理的描述的起源高和低亲和力的EGF结合位点，可以在未来的实验中进行测试。
We have examined the rotational mobility of SL-EGF, a bifunctional adduct of bis(sulfo-N-succinimidyl)-[N-15,H-2(16)]-doxyl-2-spiro-4'-pimelate and [Lys3,Tyr22]-murine epidermal growth factor, bound to the EGF receptor in A431 membrane vesicles. The linear EPR spectrum indicated that there was essentially no free SL-EGF in the bound complex preparation. To better define the rotational mobility of the SL-EGF bound to the EGF receptor, ST-EPR spectra were obtained at multiple Zeeman field modulation frequencies. Global analysis with a uniaxial rotational diffusion model of the ST-EPR data yielded two minima that have differences in rotational mobility and in orientation of the SL-EGF relative to the membrane normal axis. The rotational mobilities of the two rotational species are consistent with monomers and dimers or somewhat larger oligomers, such as trimers or tetraniers, arguing against a role for higher order receptor clustering in receptor activation. Considering the two minima and previous observations that A431 membrane vesicles contain two distinguishable ligand-binding populations, the ST-EPR spectra were fit with a model having two uniaxial rotating species. This yielded two components that were similar to those obtained from the two original one-component fits, either fast or slow rotational mobility, with different orientations. The model-dependent results obtained suggest that there are potential conformational and rotational differences in the two populations and provide a plausible description for the origin of high- and low-affinity EGF-binding sites that can be tested in future experiments.