RECEPTOR FOR GP96 ON MACROPHAGES AND DENDRITIC CELLS

巨噬细胞和树突细胞上的 GP96 受体

• 批准号: 6377711
• 负责人: Pramod K. Srivastava
• 金额: $ 25.83万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377711
• 关键词: antigen presenting cell; binding proteins; chromatography; dendritic cells; differential display technique; electron microscopy; heat shock proteins; laboratory mouse; laboratory rabbit; laboratory rat; macrophage; molecular chaperones; protein sequence; protein structure function; protein transport; receptor expression

DESCRIPTION: (Adapted from applicant's abstract) The heat shock protein gp96 forms non-covalent complexes with peptides. Gp96-peptide complexes, upon immunization, elicit peptide-specific MHC I-restricted CD8+ T lymphocyte responses. The high efficiency of this process (as little as I ng peptide complexed to 10 ug gp96 is sufficient) and the strigent requirement of professional antigen presenting cells (APCs) for the immunogenicity of gp96-peptide complexes led to the suggestion that APCs possess receptors for gp96 (gp96R). Gp96 bindings to the surface of APCs but not other cells in a saturable and competable manner and is internalized by APCs through receptor-mediated uptake by clathrin-coated pits. A 75kda gp96-binding molecule expressed on the surface of APCs but not other cells has been identified as a candidate gp96R. Uptake of gp96-bound peptides through gp96R leads to re-presentation of the peptides by MHC I of the APCs. The application aims to identify and characterize gp96R and its gene(s) and plans to carry out: 1. Identification and characterization of gp96-binding proteins on the surface of APCs. * Cross-linking of gp96 with gp96-binding proteins from APCs using reversible cross-linkers; * Chromatography of surface proteins of APCs over immobilized gp96 affinity columns; * Generation of monoclonal antibodies to gp96R using inhibition of re-presentation as assay; * Purification and characterization of gp96R. 2. Identification and characterization of transcripts specific for APCs which can re-present gp96-chaperoned peptides: * Subtraction of cDNA gp96R-cells from that of gp96R+cells; * Use of differential display to isolate gp96R transcripts; * Screening of expression library from gp96R+cells by gp96; 3. Determination if gp96,hsp90, hsp70 and Calreticulin share receptors on APCs: *Competition among the four HSPs for binding to APCs and re-presenting chaperoned peptides; 4. Characterization of the intracellular pathway through which gp96-peptide complexes traffic within the APC: *Ultrastructural analysis where gp96 and the peptide are tagged with different indicator moieties and the fate of each is followed through electron microscopy; *Analysis of epitope-flanking sequences which affect the processing and processing and presentation of gp96-chaperoned peptides in a manner that is different from that of un-chaperoned peptides.

描述：（改编自申请人的摘要）热休克蛋白GP96 与肽形成非共价复合物。 gp96肽复合物 免疫，引起肽特异性MHC I限制的CD8+ T淋巴细胞 回答。此过程的高效率（尽可能少 复合至10 ug gp96就足够了） 专业抗原呈递细胞（APC），用于免疫原性 GP96肽复合物导致了以下建议：APC拥有受体 GP96（GP96R）。 GP96与APC表面的结合，但没有其他细胞 饱和且有能力的方式，由APC通过 受体介导的涂有网格蛋白涂层的凹坑的摄取。一个75KDA GP96结合分子 在APC的表面上表示，但没有其他细胞被确定为 候选人GP96R。通过GP96R摄取GP96结合的肽会导致 APC的MHC I对肽的重新表达。该申请旨在 识别和表征GP96R及其基因并计划执行： 1。表面上GP96结合蛋白的鉴定和表征 APC。 *使用可逆的APC与GP96结合蛋白的GP96交联 交联链接； * APC的表面蛋白质色谱法上固定的GP96亲和力 列； *使用抑制对GP96R的单克隆抗体产生 重新表达为测定； * GP96R的纯化和表征。 2。识别和表征对APC特有的成绩单 可以重新促进GP96冠状肽： *从gp96r+细胞的cDNA gp96r细胞的减去； *使用微分显示器隔离GP96R转录本； *通过GP96从GP96R+细胞中筛选表达库； 3。确定是否GP96，HSP90，HSP70和钙网蛋白共享APC上的受体： *四个HSP之间的竞争与APC结合并重新呈现 伴侣肽； 4。细胞内途径的表征GP96肽通过 复合物在APC中的流量： *超微结构分析，其中gp96和肽用不同的标记 指标部分和每个指示器的命运都遵循电子 显微镜； *分析影响加工和处理的表位频率序列 GP96培养肽的加工和表现方式是 不同于未围肽的肽。