ANTIGEN BINDING, CROSSLINKING & TRANSMEMBRANE SIGNALING

抗原结合、交联

• 批准号: 3133522
• 负责人: DAVID A HOLOWKA
• 金额: $ 18.64万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1996-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3133522
• 关键词: antiantibody; antibody receptor; biological signal transduction; crosslink; erythrocytes; fluorescence spectrometry; immunoglobulin E; protein tyrosine kinase; receptor binding; tissue /cell culture

A process of fundamental importance in immunology is the antigen-mediated crosslinking of cell surface receptors that leads to transmembrane signaling in specialized cells. The critical molecular features of the receptor crosslinking events required to initiate cell activation and the biochemical events that immediately follow are not well understood. The proposed studies advance efforts toward elucidating these events and will build upon a strong foundation of methodologies and concepts from previous studies in this laboratory. These studies will focus on what has proven to be an excellent model system: the high affinity receptor for immunoglobulin E (Fc-epsilon-RI) on RBL-2H3 cells, a mucosal mast cell line. Antigen binding and crosslinking studies will assess a working model that emphasizes the dynamics of crosslinking: maximal responses occur under conditions where antigen-mediated crosslinks are rapidly breaking and reforming such that new receptors are continually being incorporated into the crosslinked aggregate. The model also explains that the reason why most bivalent ligands, which readily crosslink IgE-receptor complexes, do not stimulate cells is because they tend to form highly stable cyclic dimers. The generality of both of these aspects will be examined with a variety of bivalent ligands (including anti-IgE monoclonal antibodies) by comparing detailed binding measurements with assays of biological activity. The possible importance of receptor-receptor orientation will be investigated with measurements of resonance energy transfer. Comparative experiments with a specially developed trivalent ligands and monovalent IgE will allow detailed examination of the relative efficacy of linearly crosslinked receptor aggregates and branched networks of aggregated receptors. Studies of the early events of receptor-mediated cell activation will investigate mechanisms for the initiation and control of the signal transduction cascade. Part of these studies will focus on the central role of tyrosine kinase activation and effects of its inhibition on other pathways. Subcellular preparations will be developed to examine the mechanisms of receptor-mediated tyrosine phosphorylation of cellular and exogenous substrates, and these systems will also be analyzed with steady state kinetics in conjunction with antigen binding measurements. Other studies will examine antigen-mediated desensitization of Fc-epsilon-RI which appears to involve serine and threonine phosphorylation of a receptor subunit and also an unidentified receptor-associated protein. The desensitizing process can be reversed in a time-dependent manner by addition of a monovalent ligand that prevents further antigen-mediated crosslinking of receptors. This process appears to represent a type of immunological memory, and the molecular basis for its mechanism will be examined in conjunction with the antigen binding measurements.

免疫学中基本重要性的过程是抗原介导的 导致跨膜的细胞表面受体的交联 在专用细胞中发出信号。 临界分子特征 受体交联事件引发细胞激活和 立即随之而来的生化事件尚不清楚。 这 拟议的研究促进了阐明这些事件的努力，并将 以强大的方法和概念为基础 该实验室的先前研究。 这些研究将集中于什么 事实证明是一个出色的模型系统：高亲和力受体 对于RBL-2H3细胞上的免疫球蛋白E（FC-EPSILON-RI），粘膜桅杆 细胞系。 抗原结合和交联研究将评估一个工作模型 强调交联的动态：最大反应发生在下面 抗原介导的交联的条件迅速破裂，并且 改革使得新受体不断被纳入 交联聚集。 该模型还解释了原因 大多数二价配体很容易交叉IgE受体复合物， 不刺激细胞是因为它们倾向于形成高度稳定的环状 二聚体。 这两个方面的一般性将通过 各种二价配体（包括抗IgE单克隆抗体） 比较详细的结合测量与生物学测定 活动。 受体受体取向的可能性将 通过测量共振能量传递进行研究。 与特殊三价配体的比较实验， 单价IGE将允许详细检查相对功效 线性交联的受体聚集体和分支网络 聚集的受体。 对受体介导的细胞激活的早期事件的研究将 研究信号启动和控制的机制 转导级联。 这些研究的一部分将重点放在中央 酪氨酸激酶激活及其抑制作用的作用 途径。 将开发亚细胞制剂以检查 受体介导的酪氨酸磷酸化的机理，细胞和 外源底物，这些系统也将以稳定的 状态动力学与抗原结合测量结合。 其他 研究将检查FC-EPSILON-RI的抗原介导的脱敏 似乎涉及丝氨酸和苏氨酸的磷酸化 受体亚基以及未鉴定的受体相关蛋白。 脱敏过程可以通过时间依赖的方式逆转 添加单价配体，可防止进一步的抗原介导 受体的交联。 这个过程似乎代表了 免疫记忆和其机制的分子基础将是 与抗原结合测量结果一起检查。