Role of the Sec61 Translocon in EGF Receptor Trafficking and Signaling

这项申请旨在探索一条生长因子在细胞内运输的新途径- 激活EGF受体至胞核。此外，一种临床相关的EGF受体的能力 促进EGF受体核定位的抗体将被研究。简而言之， 初步数据显示，在加入EGF后，EGF受体缓慢地被运输到 内质网(ER)。在内质网中，这些受体与Sec61转运子相互作用 并作为非膜结合分子逆行转运到细胞质中。HSP70是 逆转转运到细胞质所必需的，并可能通过结合 可溶性EGF受体的跨膜区，以防止聚集。击倒 第61节？阻止EGF依赖的受体移位到细胞核和 Cyclin D1的表达，表明内质网易位是核定位的先兆 和EGF受体的信号功能。在前两个目标中，机械的问题是 阐述了这一途径的细胞生物学和生物化学。在最后两个目标中， 从生长控制和一种临床使用的表皮生长因子抗体方面探索了这一途径。 受体。 第一个目标是调查细胞内的运输路线， 激活的EGFR被传输到急诊室。人们建议进行实验，以确定 细胞内化机制、高尔基体和内小体可能参与这一途径 第二个目标集中在受体被Sec61识别的机制上 急诊室里的易位基因。这将测试激活的受体被识别的可能性 内质网中错误折叠的蛋白质对SEC-61依赖的已知机制 逆转易位和胞质降解。此外，这一目标将包括进行实验，以确定 参与逆转易位的细胞质和内质网因子，易位的命运(S) 受体，以及在内质网和细胞核中发现的EGF受体片段的身份。这 包括HSP70和p97，这是一种ATPase，在添加 EGF。 第三个目的将确定Sec61在表皮生长因子刺激的G1中的可能作用？？S 过渡。最终目的是探索临床上使用表皮生长因子的机制。 受体抗体C225(Erbitux)能够诱导受体转运到内质网和细胞核。 此外，还将研究Sec61在细胞对C225反应中的作用。这项拨款的重点是EGF受体，这是一种蛋白质，是几个 被批准用于临床治疗不同癌症的化疗药物。应用程序 建议研究这种受体对其正常激活物--一种激素样生长的反应 刺激细胞增殖的因子，以及对受体的抗体，它是 临床批准的治疗癌症的试剂。

This application seeks to explore a new route of intracellular trafficking of growth factor- activated EGF receptor to the nucleus. Also, the capacity of a clinically relevant EGF receptor antibody to promote nuclear localization of the EGF receptor will be investigated. In brief, preliminary data show that following the addition of EGF, EGF receptors are slowly trafficked to the endoplasmic reticulum (ER). In the ER these receptors interact with the Sec61 translocon and are retrotranslocated to the cytoplasm as non-membrane bound molecules. HSP70 is required for retrotranslocation to the cytosol and likely functions by associating with transmembrane domains of the soluble EGF receptor to prevent aggregation. Knockdown of Sec61? prevents the EGF-dependent translocation of its receptor to the nucleus and the expression of cyclin D1, indicating that ER translocation is a precursor for nuclear localization and signaling function of the EGF receptor. In the first two aims, mechanistic questions are addressed regarding the cell biology and biochemistry of this pathway. In the last two aims, the pathway is explored in terms of growth control and a clinically used antibody to the EGF receptor. The first aim proposes to investigate the intracellular trafficking route by which the activated EGFR is trafficked to the ER. Experiments are proposed to determine how different cell internalization mechanisms, the Golgi, and endosomes may participate in this pathway The second aim focuses on the mechanism by which the receptor is recognized by the Sec61 translocon in the ER. This will test the possibility that the activated receptor is recognized by the known mechanism in the ER that sorts misfolded proteins for Sec-61-dependent retrotranslocation and cytosolic degradation. Also, this aim will include experiments to identify cytoplasmic and ER factors that participate in retrotranslocation, the fate(s) of translocated receptor, and the identity of EGF receptor fragments found in the ER and nucleus. This includes HSP70 and p97, an ATPase that is tyrosine phosphorylated following the addition of EGF. The third aim will determine the possible role of Sec61 in the EGF-stimulated G1?S transition. The final aim will explore the mechanism by which the clinically employed EGF receptor antibody C225 (Erbitux) is able to induce receptor trafficking to the ER and nucleus. The role of Sec61 in cell responses to C225 will be investigated also. The focus of this grant is the EGF receptor, a protein that is a rational target for several chemotherapeutic drugs that are approved for clinical use for different cancers. The application proposes investigation of this receptor in response to its normal activator, a hormone-like growth factor that stimulates cells to proliferate, as well as an antibody to the receptor that is one of the clinically approved reagents for cancer treatment.