Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3
Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3
复制标题
DOI：
10.1074/jbc.273.13.7717
复制
发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Daly, RJ
中科院分区：
文献类型：
--
作者：
Fiddes, RJ;Campbell, DH;Daly, RJ
使用调蛋白介导的特定 erbB 受体组合的激活作为模型系统来研究 erbB3 和 erbB4 与接头蛋白生长因子受体结合 (Grb)7 的相互作用。在人乳腺癌细胞系中，在调蛋白刺激后检测到 Grb7 与两种受体的免疫共沉淀。这种关联是直接的，并由 Grb7 Src 同源 (SH)2 结构域介导。 erbB2 与 erbB3 点突变体的共表达用于绘制 Grb7 结合位点图谱。这证明酪氨酸1180和1243分别代表Grb7相互作用的主要和次要位点。尽管这些识别序列在相对于磷酸酪氨酸的 +2 处具有 Asn 残基，因此代表潜在的 Grb2 结合位点，但磷酸肽竞争和“下拉”实验表明，相对于 Grb2 SH2 结构域，它们优先与 Grb7 相互作用。取代分析表明，+3 处的精氨酸残基可以作为选择性决定因素，但其效果取决于上下文。因此，Grb2 和 Grb7 SH2 结构域具有重叠但不同的特异性。因此，这些研究将 Grb7 确定为 erbB3 和 erbB4 的体内靶标，并为 erbB3 招募 Grb7 而不是 Grb2 的能力提供了潜在机制，这是 erbB 受体中独特的特性。
Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Coexpression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.