Elucidation of Molecular Mechanism of Signal Transduction Mediated by Growth Factor-Receptor System

我们构建了一个计算机程序系统，通过核磁共振测定蛋白质的高分辨率溶液结构。heregulin是ErbB4受体的配体，与EGF受体相似，但在中枢神经系统再生和肿瘤形成中起重要作用。其结构与EGF非常相似，我们预计heregulin也具有与EGF相似的结合表面。假定的受体结合表面上的斑块与EGF完全不同，支持这些配体只与同源受体结合。我们还确定了IGF-II的溶液结构。IGF-II类似于胰岛素和IGF-I。我们发现胰岛素/IGF-I受体和IGF-II受体的结合位点位于IGF-II的相反表面。我们将结构研究扩展到细胞质状态下的蛋白质相互作用。SH2和SH3是集成在信号转导系统中的结构模块。SH2通过与受体中的磷酸酪氨酸残基结合而与活化的受体结合。Src-homology 3 （SH3）结构域通过与参与信号转导系统的靶蛋白中富含脯氨酸的基序结合介导信号转导。我们已经确定了PLCgamma和Grb2的SH3结构域的高分辨率结构。SH3结构域的三维结构非常相似，尽管序列同源性较低（20%）。但是，由于富含脯氨酸的肽对这些SH3结构域的亲和力较低，我们无法确定其复杂结构。幸运的是，从Sos衍生的VPPPVPPRRR肽以高亲和力结合到Grb2 N-SH3上，我们已经确定了该复合物的高分辨率NMR结构。核磁共振数据表明，该肽具有左旋型脯氨酸II型螺旋结构，并与SH3结构域上的三个主要位点相互作用。结合肽的取向与Abl、Fyn和p85的SH3结构域结合的富含脯氨酸的肽的取向相反。

We constructed a computer program system to determine the high reolution solution structure of proteins by NMR.The program were applied to determine the structure of heregulin, which is a ligand to ErbB4 receptor which is similar to EGF receptor but is important for regemeration of central nervous system and tumor fotmation. The structure is quite similar to EGF and we expect that heregulin also has similar binding surface to EGF.The patch on the putative receptor binding surface is quite different from that of EGF,supporting that these ligand bind exclusively to cognate receptors. We also determind the solution structure of IGF-II.IGF-II is similar to insulin and IGF-I.We found that the binding sites for insulin/IGF-I receptor and IGF-II receptor are located on the opposite surface of IGF-II.We extended our structural study to the protein interaction in the cytosolic state. SH2 and SH3 are structural module integrated in the signal transduction system. SH2 binds to the activated receptors by the binding to a phosphotyrosine residue in the receptor. Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins involved in signal transduction system. We have determined the high-resolution structure of the SH3 domains of PLCgamma and Grb2. The three dimensional structure of SH3 domains are quite similar in spite of the low sequence homology (20%). But, owing to low affinity of proline-rich peptide to those SH3 domains, we could not determind the complex structures. Fortunately, VPPPVPPRRR peptide derived from Sos bound to Grb2 N-SH3 with high affinity and we have determined the high-resolution NMR structure of the complex. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.