CD28 Triggering

CD28触发

• 批准号: 7963606
• 负责人: JIM F Miller
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7963606
• 关键词: Abbreviations; Accounting; Amino Acids; Antigen-Presenting Cells; Binding Proteins; Biological Assay; CD28 gene; Cell membrane; Disulfides; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; ITAM; Integrins; Left; Ligand Binding; Link; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Mutation; Phosphorylation; Phosphotransferases; Plant Leaves; Proteins; Recruitment Activity; Regulation; Research; Rest; Role; Signal Transduction; Signaling Molecule; T cell regulation; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TCR Activation; Tail; Testing; dimer; immunological synapse; interest; pathogen; public health relevance

DESCRIPTION (provided by applicant): The two signal model and the concept of costimulation are well engrained in our understanding of T cell regulation. It is well established that costimulation through CD28 can have a dramatic effect on T cell activation, survival, tolerance, and differentiation. However, in spite of considerable interest and effort, the regulation and mechanism of CD28 costimulation are still poorly understood. CD28 signaling is mediated through the recruitment of cytosolic signaling molecules to specific motifs within the cytosolic tail (CT) domain of CD28. In an effort to understand how ligand binding to the lumenal domain of CD28 could transduce changes to the cytosolic domains to initiate signaling, we established a FRET assay. CFP and YFP were fused to the end of the CT of CD28. CD28 is a disulfide-linked dimer and when CD28-CFP and CD28-YFP were co-expressed, high level of FRET was directed, indicating that the ends of the CT domains are in close proximity within the CD28 dimer. When CD28 was recruited to the immunological synapse, the level of FRET was reduced, indicating that there was some structural change in the orientation of the CT domains within the CD28 dimer. Surprisingly, this change in FRET was not mediated by CD28 ligand binding, but instead was mediated through TCR signaling, even in the absence of CD28 ligand binding. This suggests that TCR signaling can activate CD28, possibly impacting on ligand binding (in analogy to inside-out signaling for integrin activation), immunological synapse recruitment, or signal transduction. The overall goal of this R21 application is to identify the mechanism and functional consequence of this TCR- mediated change in the orientation of the CD28 CT domains. We will do this through two Specific Aims. 1, Determine the molecular events associated with TCR induced changes in the orientation of the CD28 CT domains. 2, Determine whether TCR signaling regulates CD28 ligand binding. PUBLIC HEALTH RELEVANCE: T cell activation requires the specific recognition of pathogen-specific proteins by the TCR and co-signaling through costimulatory molecules, most notably CD28. Although the function of CD28 is well described, the molecular events associated with CD28 function as not well understood. We have made the surprising finding that the TCR may modify the conformation of CD28 and so regulate the function of CD28.

描述（由申请人提供）：两个信号模型和共刺激的概念完全介入了我们对T细胞调节的理解。众所周知，通过CD28进行共刺激会对T细胞激活，生存，耐受性和分化产生巨大影响。然而，尽管有很大的兴趣和努力，但CD28共刺激的调节和机制仍然很少了解。 CD28信号通过募集胞质信号分子募集到CD28的胞质尾部（CT）结构域内的特定基序。为了了解配体与CD28的腔内结构域的结合如何将变化转换为胞质域以启动信号传导，我们建立了一个FRET分析。 CFP和YFP融合到CD28 CT的末端。 CD28是一种二硫化合物二聚体，当CD28-CFP和CD28-YFP共表达时，指向高水平的FRET，表明CT结构域的末端在CD28二聚体内紧邻。当将CD28募集到免疫突触时，降低了FRET的水平，表明CD28二聚体内CT域的方向存在一些结构性变化。令人惊讶的是，这种FRET的这种变化不是由CD28配体结合介导的，而是通过TCR信号传导介导的，即使在没有CD28配体结合的情况下也是通过TCR信号传导介导的。这表明TCR信号传导可以激活CD28，可能会影响配体结合（类似于整联蛋白激活的内而外信号传导），免疫突触募集或信号转导。该R21应用程序的总体目标是确定CD28 CT域的方向变化的机制和功能后果。我们将通过两个具体的目标来做到这一点。 1，确定与TCR诱导的CD28 CT域方向变化相关的分子事件。 2，确定TCR信号是否调节CD28配体结合。 公共卫生相关性：T细胞激活需要通过TCR对病原体特异性蛋白的特定识别，并通过共刺激分子（最著名的CD28）共同信号。尽管很好地描述了CD28的功能，但与CD28相关的分子事件效果不佳。我们已经令人惊讶的是，TCR可能会改变CD28的构象，从而调节CD28的功能。