MOLECULAR MECHANISMS OF ENDOSOME FUSION

我们的目标是明确早期内吞途径中的内化和分选机制。 这些机制涉及重要的生理过程，如葡萄糖转运和生长因子 受体下调，分别在糖尿病和癌症等疾病中被破坏。 通过其FYVE结构域与磷脂酰肌醇3-磷酸（PI 3 P）相互作用的蛋白质在细胞内起重要作用。 但不完全理解的作用内吞运输。在上一个资助期内， 详细了解EEA 1与表面结合的结构和细胞基础 早期内体通过其FYVE结构域与PI 3 P的相互作用，但仍存在许多问题 关于PI 3 P、EEA 1和其他含有FYVE结构域的蛋白在哺乳动物中的精确功能， 内吞运输我们已经发现EEA 1的缺失深刻地抑制EGF受体（EGFR）， 降解，但对转铁蛋白内化和再循环的影响可忽略不计。这些新结果 将EEA 1置于与转运货物（如EGFR）相关的内吞途径中的一个点， 多泡体（MVB）/溶酶体途径，并表明其他含有FYVE结构域 Rabenosyn-5等蛋白质可能与转铁蛋白等蛋白质的再循环更相关 受体和GLUT 4的质膜。此外，我们还实现了直接可视化， 在活细胞中真实的时间的荧光配体结合和内化，以及GT3的激活 Rab 5和EEA 1对EGF应答的早期内体的募集。与项目合作 1，3和4，以及与成像核心，我们将使用结构，生化和成像的组合 技术来测试以下假设：1：EGF的高和低亲和力结合是由于 EGFR在不同质膜亚结构域中的动态分布。2：激活， 泛素化EGFR通过与含有Vps 9结构域的蛋白相互作用激活Rab 5， 包括释放RabexS的自身抑制的机制。3：EEA 1与激活的RabS相互作用， EGFR通过MVB-溶酶体途径降解所需。4：TfR和GLUT 4的分类， EGFR依赖于Rabs 4和5的相对活性，以及它们与EGFR的相互排斥作用。 双重PI 3 P-Rab结合蛋白EEA 1、Rabenosyn和Rabip 4。

Our goal is to define the mechanisms of internalization and sorting in the early endocytic pathway. These mechanisms relate to important physiological processes, such as glucose transport and growth-factor receptor down regulation, which are disrupted in diseases such as diabetes and cancer, respectively. Proteins that interact with phosphatidylinositol 3-phosphate (PI3P) via their FYVE domain play an important yet incompletely understood role in endocytic trafficking. During the previous funding period we achieved a detailed understanding of the structural and cellular basis for the association of EEA1 to the surface of early endosomes through the interaction of its FYVE domain to PI3P, but many questions remain unanswered regarding the precise function of PI3P, EEA1 and other FYVE-domain containing proteins in endocytic trafficking. We have found that depletion of EEA1 profoundly inhibits EGF receptor (EGFR) degradation, yet has a negligible effect on transferrin internalization and recycling. These new results place EEA1 at a point in the endocytic pathway related to the trafficking of cargo such as the EGFR towards the multivesicular body (MVB) /lysosomal pathway, and suggest that other FYVE-domain containing proteins, such as Rabenosyn-5, may be more relevant for recycling of proteins such as the transferrin receptor and GLUT4 to the plasma membrane. Moreover, we have achieved the direct visualization of fluorescent ligand binding and internalization in live cells in real time, as well as the activation of GTPase Rab5 and the recruitment of EEA1 to early endosomes in response to EGF. In collaboration with Projects 1,3 and 4, as well as with the Imaging Core, we will use a combination of structural, biochemical and imaging technologies to test the following hypotheses: 1: That high and low affinity binding of EGF is due to the dynamic distribution of EGFRs among distinct plasma membrane sub domains. 2: That activated, ubiquitinated EGFR activates Rab5 through its interaction with Vps9 domain-containing proteins, and by mechanisms that include release of autoinhibition of RabexS. 3: That EEA1 interacting with activated RabS is required for EGFR degradation via the MVB-lysosomal pathway. 4: That sorting of the TfR and GLUT4 from EGFR depends on the relative activities of Rabs 4 and 5, and their mutually exclusive interactions with the dual PI3P-Rab binding proteins EEA1, RabenosynSand Rabip4.