Role of the Sec61 Translocon in EGF Receptor Trafficking and Signaling

描述（由申请人提供）：本申请旨在探索生长因子激活的EGF受体到细胞核的细胞内运输的新途径。此外，将研究临床相关的EGF受体抗体促进EGF受体核定位的能力。简而言之，初步数据表明，在加入EGF后，EGF受体被缓慢地转运到内质网（ER）。在内质网中，这些受体与Sec61易位子相互作用，并作为非膜结合分子逆转录到细胞质中。HSP70是细胞质逆转录所必需的，并且可能通过与可溶性EGF受体的跨膜结构域结合来防止聚集。击倒Sec61？阻止EGF依赖性受体向细胞核的易位和cyclin D1的表达，表明内质网易位是EGF受体核定位和信号功能的前体。在前两个目标中，解决了有关该途径的细胞生物学和生物化学机制问题。在最后两个目标中，从生长控制和临床使用的EGF受体抗体的角度探讨了该途径。

DESCRIPTION (provided by applicant): 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.