Establishing the Molecular Mechanisms of BCR Endocytosis

描述(申请人提供)：B细胞受体(BCR)介导的抗原内化是抗原处理和提呈途径的第一步，这一现象对于同源B细胞和T细胞的相互作用、B细胞的激活和免疫记忆的建立是必不可少的。然而，抗原-BCR(Ag-BCR)复合体内化的细胞和分子机制仍然不清楚，以至于甚至BCR胞浆结构域中的内吞基序的同一性也基本上还不清楚。因此，该方案的总体目标是建立不同BCR连接和交联条件下的Ag-BCR内化机制，并确定Ag-BCR内吞作用途径的调节如何影响BCR介导的抗原处理和递呈等重要事件。根据该实验室和其他实验室已发表的工作，我们假设Ag-BCR的内化通过两个不同的内吞网关发生，即网状蛋白包裹的凹坑和质膜脂筏，这两个不同的网关的参与水平取决于BCR交联度和BCR信号诱导的BCR内吞基序活性的变化。此外，我们认为，这些通路在Ag-BCR内化中的相对作用的调节将影响抗原处理和递呈的机制。为了验证这一假说，我们将对天然膜进行EM分析，以确定在不同的BCR连接条件下，通过质膜网状蛋白包裹的凹坑和脂筏的Ag-BCR内化水平(目标1)，鉴定和表征BCR胞浆尾部的内吞基序(S)，并确定信号诱导的BCR磷酸化对内吞基序活性的影响(目标2)，并建立内化对Ag-BCR泛素化(本实验室最近报道的一种新现象)的影响机制以及内化的Ag-BCR复合体的运输、加工和呈现(目标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、加工和递呈的细胞和分子机制。此外，应用这些发现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.