Inhibition of hGrb10 binding to the insulin receptor by functional domain-mediated oligomerization
Inhibition of hGrb10 binding to the insulin receptor by functional domain-mediated oligomerization
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DOI：
10.1074/jbc.273.28.17720
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发表时间：
1998-07-10
影响因子：
4.8
通讯作者：
Liu, F
中科院分区：
文献类型：
--
作者：
Dong, LQ;Porter, S;Liu, F
hGrb 10是一种新发现的含Src同源2（SH 2）和pleckstrin同源（PH）结构域的蛋白质，其结合自磷酸化受体酪氨酸激酶，包括胰岛素和胰岛素样生长因子受体。为了鉴定与hGrb 10相互作用的潜在下游蛋白，我们使用全长hGrb 10 γ作为诱饵筛选酵母双杂交cDNA文库。发现hGrb 10的片段，其包括IPS（插入在PH和SH 2结构域之间）和SH 2结构域，以高亲和力结合全长蛋白。通过体外谷胱甘肽S-转移酶融合蛋白结合研究进一步证实了IPSI SH 2结构域和全长hGrb 10之间的相互作用。凝胶过滤分析表明，hGrb 10在哺乳动物细胞中经历了四聚化。这种相互作用涉及至少两个功能结构域，IPS/SH 2区和PH结构域，它们都与hGrb 10 γ的NH 2-末端氨基酸序列相互作用（hGrb 10 γ Δ C，残基4-414），竞争性研究显示hGrb 10 γ Δ C抑制hGrb 10与酪氨酸磷酸化胰岛素受体的结合，提示该区域可能在hGrb 10/胰岛素受体相互作用中起调节作用。我们提出了一个模型hGrb 10四聚体和其在受体酪氨酸激酶信号转导的潜在作用。
hGrb10 is a newly identified Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing protein that binds to autophosphorylated receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors. To identify potential downstream proteins that interact with hGrb10, we screened a yeast two-hybrid cDNA library using the full-length hGrb10 gamma as bait. A fragment of hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein. The interaction between the IPSI SH2 domain and the full-length hGrb10 was further confirmed by in, vitro glutathione S-transferase fusion protein binding studies. Gel filtration assays showed that hGrb10 underwent tetramerization in mammalian cells. The interaction involved at least two functional domains, the IPS/SH2 region and the PH domain, both of which interacted with the NH2-terminal amino acid sequence of hGrb10 gamma (hGrb10 gamma Delta C, residues 4-414), Competition studies showed that hGrb10 gamma Delta C inhibited the binding of hGrb10 to the tyrosine-phosphorylated insulin receptor, suggesting that this region may play a regulatory role in hGrb10/insulin receptor interaction. We present a model for hGrb10 tetramerization and its potential role in receptor tyrosine kinase signal transduction.