Establishing the Molecular Mechanisms of BCR Endocytosis

描述（由申请人提供）：B细胞受体（BCR）介导的抗原内化是抗原加工和呈递途径的第一步，这种现象对于同源B细胞-T细胞相互作用、B细胞激活和免疫记忆的建立至关重要。然而，抗原-BCR (Ag-BCR) 复合物内化的细胞和分子机制仍然不明确，甚至 BCR 细胞质域内的内吞基序的身份也基本上没有被表征。因此，该提案的总体目标是建立不同BCR连接和交联条件下Ag-BCR内化的机制，并确定Ag-BCR内吞作用途径的调节如何影响BCR介导的抗原加工和呈递等重要事件。根据该实验室和其他实验室发表的工作，我们假设 Ag-BCR 内化通过两个不同的内吞网关（网格蛋白包被的凹坑和质膜脂筏）发生，并且这两个不同网关的参与水平取决于 BCR 交联的水平和 BCR 信号诱导的 BCR 内吞基序活性变化。此外，我们认为调节这些网关在 Ag-BCR 内化中的相对作用将影响抗原加工和呈递的机制。为了检验这一假设，我们将对天然膜进行 EM 分析，以确定不同 BCR 连接条件下通过质膜网格蛋白包被的凹坑和脂筏的 Ag-BCR 内化水平（目标 1），识别和表征 BCR 细胞质尾部内的内吞基序，确定信号诱导的 BCR 磷酸化对内吞基序活性的影响（目标 2），并建立内化机制对内吞基序的影响。 Ag-BCR 泛素化（我们实验室最近报道的一种新现象）以及内化 Ag-BCR 复合物的细胞内运输、加工和呈递（目标 3）。成功完成这些研究将有助于更好地了解 BCR 介导的抗原内化、加工和呈递的细胞和分子机制。此外，这些发现的应用将有助于开发改进的疫苗和控制自身免疫的方法。

DESCRIPTION (provided by applicant): B cell receptor (BCR)-mediated antigen internalization is the first step in the pathway of antigen processing and presentation, a phenomenon which is essential for cognate B cell-T cell interactions, B cell activation and establishment of immunological memory. However, the cellular and molecular mechanisms underlying the internalization of antigen-BCR (Ag-BCR) complexes remain ill defined, so much so that even the identity of the endocytosis motifs within the cytoplasmic domains of the BCR remain essentially uncharacterized. Therefore, the overall goals of this proposal are to establish the mechanisms of Ag-BCR internalization under different conditions of BCR ligation and cross-linking, and determine how modulation of the pathway of Ag-BCR endocytosis impacts important events such BCR-mediated antigen processing and presentation. Based on published work from this and other laboratories, we hypothesize that Ag-BCR internalization occurs via two distinct endocytic gateways, clathrin coated pits and plasma membrane lipid rafts, and that the level of engagement of these two distinct gateways depends upon the level of BCR cross-linking and BCR signaling-induced changes in the activity of BCR endocytosis motifs. Moreover, we propose that modulation of the relative roles of each of these gateways in Ag-BCR internalization will affect the mechanism of antigen processing and presentation. To test this hypothesis we will perform EM analysis of native membranes to determine the level of Ag-BCR internalization via plasma membrane clathrin coated pits and lipid rafts under different conditions of BCR ligation (Aim 1), identify and characterize the endocytosis motif(s) within the cytoplasmic tail of the BCR and determine the impact of signaling-induced BCR phosphorylation on endocytosis motif activity (Aim 2), and establish the impact of the mechanism of internalization on Ag-BCR ubiquitination (a novel phenomenon recently reported by our laboratory) as well as the intracellular trafficking, processing and presentation of internalized Ag-BCR complexes (Aim 3). Successful completion of these studies will result in a better understanding of the cellular and molecular mechanisms of BCR-mediated antigen internalization, processing and presentation. Moreover, application of these findings will allow for the development of improved vaccines and approaches to control autoimmunity.