Control of ErbB Receptor Sorting in Endosomes

描述（由申请方提供）：内体装置是胞吞和胞吐途径中膜分选的主要部位。对于配体激活的受体，它还提供了一种机制，用于实现来自不同膜区室的细胞信号传导的适当平衡。该提案的主要目的是了解内吞作用如何控制细胞对EGF受体（EGFR）的反应，EGFR是ErbB受体家族的原型。内吞运输的复杂性预测EGFR在细胞中的不同位置使用大量不同的分选信号。这些信号的表征为理解EGFR在内体装置中转运的调控提供了框架。我们已经确定了控制EGFR运输中关键基准的三种非重叠分选信号：679-LL，其控制多泡内体至溶酶体转运中间体或MVE中的分选; 954-YLVI，其控制与外排途径交叉的位点处的溶酶体分选;和662-RxxxxPLTP，其控制从内体至质膜的分选。完成拟议的研究将提供新的见解，这些信号在配体调节的EGFR贩运的协调行动。由于相关的ErbB受体与EGFR相比具有不同的分选信号，因此我们还将深入了解ErbB信号传导一般如何受细胞内运输控制。这对于ErbB 2尤其重要，其通过EGFR的配体依赖性活化需要调节内吞后运输的EGFR序列，而不是内在酪氨酸激酶活性。在规定步骤破坏EGFR转运的显性抑制突变的可用性也提供了一组独特的试剂，可用于研究内体信号传导的时间和空间组织。以下假设将被测试：1。配体诱导的EGFR内吞后分选至溶酶体由以逐步方式起作用的多个分选信号的协同作用介导。2. 679-LL MVE分选信号被识别为更大基序的一部分，其生理功能取决于蛋白质-蛋白质相互作用。3. 662-RxxxxPLTP分选信号控制EGFR再循环至质膜，并受MAP激酶介导的Thr 669磷酸化调节。4. 679-LL分选信号通过控制胞吞作用期间引起的生长刺激途径的平衡来调节EGFR信号传导。这一假设是基于新的数据表明，与野生型相比，具有失活L 679 A，L 680 A突变的EGFR选择性地激活生存途径，并将使用体外和体内实验模型进行测试。

DESCRIPTION (provided by applicant): The endosomal apparatus is a major site of membrane sorting in both endocytic and exocytic pathways. For ligand activated receptors it also provides a mechanism for achieving the proper balance of cell signaling from different membrane compartments. A main objective of this proposal is to understand how endocytosis controls cellular responses to the EGF receptor (EGFR), the prototype for the ErbB receptor family. The complexity of endocytic trafficking predicts that EGFRs use a multitude of distinct sorting signals at different locations in the cell. Characterization of these signals provides a framework for understanding regulation of EGFR transit in the endosomal apparatus. We have identified three non-overlapping sorting signals that control critical benchmarks in EGFR trafficking: 679-LL, which controls sorting in multivesicular endosome-to-lysosome transport intermediates or MVEs; 954-YLVI, which controls lysosomal sorting at a site intersecting the exocytic pathway; and 662-RxxxxPLTP, which controls sorting from endosomes to the plasma membrane. Completion of the proposed studies will provide new insights on the coordinated action of these signals during ligand-regulated EGFR trafficking. Since related ErbB receptors have divergent sorting signals compared to EGFR, we will also gain insight to how ErbB signaling in general is controlled by intracellular trafficking. This is particularly important for ErbB2, whose ligand-dependent activation by EGFR requires EGFR sequences that regulate post-endocytic trafficking and not intrinsic tyrosine kinase activity. The availability of dominant-inhibitory mutations that disrupt EGFR trafficking at defined steps also provides a unique set of reagents with which to study the temporal and spatial organization of signaling from endosomes. The following hypotheses will be tested: 1. Ligand-induced EGFR post-endocytic sorting to lysosomes is mediated by the concerted action of multiple sorting signals acting in a step-wise fashion. 2. The 679-LL MVE sorting signal is recognized as part of a larger motif whose physiological function is dependent on protein-protein interactions. 3. The 662-RxxxxPLTP sorting signal controls EGFR recycling to the plasma membrane, and is regulated by MAP kinase-mediated phosphorylation of Thr669. 4. The 679-LL sorting signal regulates EGFR signaling by controlling the balance of growth stimulatory pathways elicited during endocytosis. This hypothesis is based on new data suggesting that EGFRs with an inactivating L679A,L680A mutation selectively activate survival pathways compared to wild-type, and will be tested using both in vitro and in vivo experimental models.