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递呈途径的第一步，是同源B细胞- t细胞相互作用、B细胞活化和免疫记忆建立所必需的现象。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，以至于BCR细胞质结构域内吞作用基序的身份仍然基本上没有被表征。因此，本研究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内吞途径的调节如何影响BCR介导的抗原加工和递呈等重要事件。基于本实验室和其他实验室发表的工作，我们假设Ag-BCR内化发生在两个不同的内吞通道，网格蛋白包被的凹窝和质膜脂筏，并且这两个不同通道的参与水平取决于BCR交联的水平和BCR信号诱导的BCR内吞基体活性的变化。此外，我们提出，这些通道在Ag-BCR内化中的相对作用的调节将影响抗原加工和递呈的机制。为了验证这一假设，我们将对天然膜进行EM分析，以确定在不同BCR连接条件下，通过质膜网格蛋白包覆的凹点和脂筏，Ag-BCR内化的水平（目的1），识别和表征BCR胞质尾部的内吞基序(s)，并确定信号诱导的BCR磷酸化对内吞基序活性的影响（目的2）。并确定内化机制对Ag-BCR泛素化的影响（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、加工和呈现（Aim 3）。这些研究的成功完成将有助于更好地理解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.