EPIDERMAL GROWTH-FACTOR BINDING INDUCES A CONFORMATIONAL CHANGE IN THE EXTERNAL DOMAIN OF ITS RECEPTOR
EPIDERMAL GROWTH-FACTOR BINDING INDUCES A CONFORMATIONAL CHANGE IN THE EXTERNAL DOMAIN OF ITS RECEPTOR
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DOI：
10.1002/j.1460-2075.1989.tb08596.x
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发表时间：
1989-12-20
期刊：
影响因子：
11.4
通讯作者：
MCDONALD, N
中科院分区：
文献类型：
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作者：
GREENFIELD, C;HILES, I;MCDONALD, N
为了研究人表皮生长因子受体胞外EGF结合域的性质，我们用含有编码亲本分子完整胞外区的cDNA的适当工程杆状病毒载体感染昆虫细胞。这导致了正确折叠的、稳定的、110kd的蛋白，具有200 nM的EGF结合亲和力。使用一系列三个标准的层析步骤，从1升细胞培养物中常规纯化出毫克量的蛋白质。用圆二色谱和荧光光谱技术研究了不同EGF配体加入前后胞外结构域的性质。对胞外结构域的远紫外CD光谱的二级结构分析表明，相当大比例的α-螺旋和β-折叠与已发表的EGF受体模型一致。受体的配基添加在近紫外圆二色谱和远紫外圆二色谱上都显示出不同，并且每个配基的添加都是相似的，这表明未络合的和复杂的受体之间的构象差异相似。稳态荧光测量表明，存在于胞外区域的色氨酸残基被掩埋，而配体中可溶于溶剂的色氨酸被掩埋在与受体的结合上。通过荧光各向异性衰变测量的受体-配体络合物的旋转相关时间从每一种情况下的6 ns减少到2.5 ns。这可能表明受体的色氨酸环境对配体结合产生了扰动。超速离心研究表明，配基加成没有发生聚集，这不能解释观察到的与CD或荧光的差异。因此，我们得出结论，测量到的差异是特定配体诱导受体和/或EGF配体构象变化的结果。
To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of .alpha.-helix and .beta.-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence. We therefore conclude that the measured differences are a result of a specific ligand-induced conformational change in the receptor and/or the EGF ligand.