Kohda D, Odaka M, Lax I, Kawasaki H, Suzuki K, Ullrich A, Schlessinger J, Inagaki F
Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Japan.
J Biol Chem. 1993 Jan 25;268(3):1976-81.
Elucidation of the three-dimensional structure of the complex of the epidermal growth factor (EGF) and its receptor is essential for understanding the molecular mechanisms of the EGF-receptor interaction and EGF-induced receptor-receptor interaction. NMR is useful to investigate interactions in solution between macromolecules at atomic resolution, but has a limitation in molecular masses of target proteins: less than 300 residues. We have prepared a fragment with apparent molecular mass of 40 kDa in SDS gels from the soluble extracellular domain of the EGF receptor (sEGFR, 619 residues) by sequential limited proteolysis with proteinase K and bromelain. This fragment is a monomeric structural domain consisting of 202 amino acid residues (Cys302-Arg503) and 18-kDa sugar chains, and binds EGF and transforming growth factor-alpha (TGF alpha). This 40-kDa domain has a dissociation constant of about 1 microM for human TGF alpha, which is similar to that of the parental sEGFR. sEGFR oligomerizes in response to EGF and TGF alpha, while the 40-kDa domain does not, suggesting that the sequences other than this domain is required for receptor oligomerization. The 40-kDa ligand-binding domain described in this report is suitable for analysis by various physico-chemical approaches such as NMR.
阐明表皮生长因子（EGF）及其受体复合物的三维结构对于理解EGF-受体相互作用和EGF诱导的受体-受体相互作用的分子机制至关重要。核磁共振（NMR）有助于在原子分辨率下研究溶液中大分子之间的相互作用，但对目标蛋白质的分子量有限制：小于300个残基。我们通过用蛋白酶K和菠萝蛋白酶进行顺序有限蛋白水解，从EGF受体（sEGFR，619个残基）的可溶性细胞外结构域制备了在SDS凝胶中表观分子量为40 kDa的片段。该片段是由202个氨基酸残基（Cys302-Arg503）和18 kDa糖链组成的单体结构域，可结合EGF和转化生长因子-α（TGFα）。这个40 kDa的结构域与人TGFα的解离常数约为1 microM，与亲本sEGFR相似。sEGFR会响应EGF和TGFα而发生寡聚化，而40 kDa的结构域则不会，这表明该结构域以外的序列是受体寡聚化所必需的。本报告中描述的40 kDa配体结合结构域适用于通过各种物理化学方法（如NMR）进行分析。
