Insulin-like Growth Factor I Receptor Signaling

胰岛素样生长因子 I 受体信号转导

• 批准号: 6946274
• 负责人: PETER NISSLEY
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 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/6946274
• 关键词: 3T3 cells; MCF7 cell; biological signal transduction; cell line; genetic library; growth factor receptors; insulinlike growth factor; mitogen activated protein kinase; monoclonal antibody; neoplasm /cancer immunotherapy; neoplastic cell; protein structure function; receptor binding; serine threonine protein kinase; transfection; yeast two hybrid system

In addition to being important for normal fetal and postnatal growth, there is increasing evidence that insulin-like growth factors I and II (IGF-I, IGF-II) also support the growth of certain cancers. Biologic responses to IGF-I and IGF-II are signaled by the IGF-I receptor. Therefore, we are focusing our research effort on understanding signaling by the IGF-I receptor. We have used the yeast two-hybrid system to identify new binding partners for the IGF-I receptor. We have inserted the cytoplasmic domain of the IGF-I receptor into the LexA DNA binding vector. Poly A mRNA was prepared from the human osteosarcoma cell line, MG-63, and given to a commercial laboratory for preparation of a cDNA library in the yeast two-hybrid activation domain vector. The library screen identified IGF-I receptor binding partners that we and others had previously identified (p85 regulatory subunit of PI3-K, p66 Shc, Grb10, 14-3-3 beta and zeta, SOCS-1, SH2-B/PSM, RACK-1) as well as new interactors (STAT5a, GIPC, Ril, and 4 clones that are not fully characterized). We are focusing on GIPC which represented 25% of the most strongly interacting clones. GIPC did not interact with the insulin receptor in the yeast two hybrid assay. GIPC is a 333 amino acid protein with a central SH2 domain. Mutational analysis in the yeast two-hybrid system showed that GIPC PDZ domain binds to the carboxy tail of the IGF-I receptor. GIPC dimerizes and the dimerization domain has been shown to include the amino terminal domain as well as a portion of the PDZ domain. IGF-I receptor constructs that have been mutated to prevent GIPC binding have been transfected into NIH 3T3 cells in order to explore the function of GIPC in IGF-I receptor signaling. Cells transfected with receptors mutated for GIPC binding did not form colonies in soft agar, suggesting that GIPC binding to the receptor may be required for a transformation pathway. We have developed a mouse monoclonal antibody (4G11) directed against the human IGF-I receptor. This monoclonal antibody blocks the binding of radiolabeled IGF-I to MCF-7 breast cancer cells and MG-63 osteosarcoma cells and downregulates the receptor. Receptor downregulation results in inhibition of IGF-I stimulated activation of Akt and MAPK in MCF-7 cells. The Fab fragment of the monoclonal antibody inhibits binding of IGF-I to the receptor but does not cause receptor downregulation, suggesting that receptor aggregation is required for downregulation. Full recovery of receptor expression following downregulation requires 48 hrs. This result suggests that intermittent treatment with the monoclonal antibody in vivo may be sufficient to block IGF-I receptor signaling in cancers.

除了对胎儿和出生后的正常生长很重要外，越来越多的证据表明，胰岛素样生长因子I和II(IGF-I，IGF-II)也支持某些癌症的生长。对IGF-I和IGF-II的生物反应是通过IGF-I受体传递信号的。因此，我们的研究重点是了解IGF-I受体所传递的信号。 我们已经使用酵母双杂交系统来确定IGF-I受体的新结合伙伴。我们已将IGF-I受体的胞质结构域插入到LexA DNA结合载体中。从人骨肉瘤细胞系MG-63中制备Poly A mRNA，并将其提供给商业实验室，以制备酵母双杂交激活结构域载体中的cDNA文库。文库筛选鉴定了我们和其他人以前鉴定的IGF-I受体结合伙伴(PI3-K的P85调节亚基、p66Shc、Grb10、14-3-3 Beta和Zeta、SoCS-1、SH2-B/PSM、Rack-1)以及新的相互作用因子(Stat5a、GIPC、rIL和4个尚未完全鉴定的克隆)。我们将重点放在GIPC上，它代表了25%的最强交互克隆。在酵母双杂交实验中，GIPC不与胰岛素受体相互作用。GIPC是一个由333个氨基酸组成的蛋白，其中心结构域为SH2。酵母双杂交系统中的突变分析表明，GIPC PDZ结构域与IGF-I受体的羧基尾部结合。GIPC二聚化，二聚化结构域被证明包括氨基末端结构域和PDZ结构域的一部分。为了探讨GIPC在IGF-I受体信号转导中的作用，我们将突变后的IGF-I受体结构导入NIH3T3细胞。与GIPC结合突变的受体的细胞在软琼脂中没有形成菌落，这表明GIPC与受体结合可能是转化途径所必需的。 我们研制了一种针对人IGF-I受体的鼠单抗(4G11)。这种单抗可以阻断放射性标记的IGF-I与MCF-7乳腺癌细胞和MG-63骨肉瘤细胞的结合，并下调受体的表达。受体下调可抑制IGF-I对MCF-7细胞Akt和MAPK的激活。单抗的Fab片段抑制IGF-I与受体的结合，但不会导致受体下调，这表明下调需要受体聚集。下调后受体表达的完全恢复需要48小时。这一结果表明，间歇性地在体内用单抗治疗可能足以阻断癌症中的IGF-I受体信号。