MECHANISM OF ACTION OF INSULIN-LIKE GROWTH FACTORS

胰岛素样生长因子的作用机制

• 批准号: 5200914
• 负责人: S P NISSLEY
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200914
• 关键词: biological signal transduction; chimeric proteins; growth factor receptors; human tissue; insulinlike growth factor; intracellular transport; lysosomes; protein transport; tissue /cell culture; transfection; western blottings

An important question concerning signaling by the insulin-like growth factor I (IGF-I) receptor is whether the downstream effector protein SHC interacts directly with the receptor or with a docking protein called insulin receptor substrate 1 (IRS-1). In collaboration with Dr. Richard Furlanetto, University of Rochester, we have employed the yeast two hybrid system to explore possible direct interactions between the IGF-I receptor and SHC. This utilizes a hybrid protein containing the bacterial LexA DNA binding domain fused to the intracellular portion of the IGF-I receptor, hybrids containing an activation domain fused to full length SHC or fragments of SHC, and a yeast strain containing LexA operator sequences fused to leu and lac Z reporter genes. When yeast were transfected with the LexA-IGF-I receptor plasmid, a fusion protein of the predicted molecular size was detected by Western blotting. This protein contained phosphotyrosine indicating that the receptor had undergone autophosphorylation. Coexpression of LexA-IGF-I receptor with activation domain-SHC hybrid resulted in activation of both reporter genes. Neither reporter gene was activated when an IGF-I receptor kinase negative mutant was used in the two-hybrid system. Surprisingly, coexpression of LexA-IGF-I receptor hybrid with activation domain SHC-SH2 domain hybrid resulted in much weaker interaction than obtained with full length SHC. This result tells us that a domain of the SHC molecule outside of the SH2 domain was largely responsible for the strong interaction of the receptor and full length SHC, and is consistent with recent reports describing interaction of the amino terminal portion of SHC with phosphotyrosine containing proteins. Studies have now been completed that test the hypothesis that endogenous IGF-II can modulate the trafficking of lysosomal enzymes by blocking binding to the IGF- II/mannose 6-phosphate receptor. MCF-7 human breast cancer cells overexpressing a precursor form of IGF-II exhibit a decreased ratio of intracellular to extracellular cathepsin D consistent with endogenous IGF-II interfering with intracellular routing of lysosomal enzymes to lysosomes and inhibition of uptake of lysosomal enzymes from the media.

胰岛素样生长信号的一个重要问题 因子I(IGF-I)受体是下游的效应蛋白 SHC直接与受体或与对接蛋白相互作用 称为胰岛素受体底物1(IRS-1)。与 罗切斯特大学的Richard Furlanetto博士，我们已经聘请了 酵母双杂交系统探索可能的直接相互作用 IGF-I受体和SHC之间的关系。这利用了一种杂交蛋白质 包含与细菌LexA DNA结合域融合的 IGF-I受体的细胞内部分，包含一个 与全长SHC或SHC片段融合的激活结构域，以及 融合Leu和lac Z的含有LexA操作符序列的酵母菌株 记者基因。LexA-IGF-I基因在酵母中的表达 受体质粒，预测的分子大小的融合蛋白是 Western blotting检测到。该蛋白质含有磷酸酪氨酸。 这表明该受体已经发生了自动磷酸化。 激活结构域-SHC杂交体共表达LexA-IGF-I受体 导致两个报告基因的激活。既不是报告基因 当使用IGF-I受体激酶阴性突变体时被激活 在双杂交系统中。令人惊讶的是，LexA-IGF-I的共表达 受体与激活结构域SHC-SH2结构域杂交 与全长SHC相比，相互作用要弱得多。这 结果表明，SHC分子在SH2外的一个结构域 域名主要是导致了 受体和全长SHC，并与最近的报道一致 描述了SHC的氨基末端部分与 含有磷酸酪氨酸的蛋白质。研究现已完成 这验证了内源性IGF-II可以调节 通过阻断与IGF-1的结合来运输溶酶体酶 II/甘露糖6-磷酸受体。人乳腺癌细胞MCF-7 过度表达IGF-II前体形式的比例降低 细胞内至细胞外组织蛋白酶D与内源性一致 IGF-II干扰溶酶体酶在细胞内的传递 溶酶体及其对溶酶体酶摄取的抑制 媒体。