Chemokine Receptor Chimeras by Synthetic Gene Library

合成基因库的趋化因子受体嵌合体

• 批准号: 7093238
• 负责人: Michael Loran Dustin
• 金额: $ 21.13万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093238
• 关键词: T cell receptor; T lymphocyte; antigen presentation; antigen presenting cell; bioengineering /biomedical engineering; biological signal transduction; biotechnology; cell cell interaction; cell migration; cell surface receptors; chemokine receptor; chemotaxis; chimeric proteins; combinatorial chemistry; genetic library; genetic screening; genetically modified animals; intracellular; laboratory mouse; ligands; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; phenotype; protein engineering; protein structure; protein structure function; receptor binding; receptor coupling

DESCRIPTION (provided by applicant): Chemokines are critical for directing the traffic and organization of immune cells. We and others have shown that T cells migrate within lymphoid organs at high speeds. Under certain conditions, when migrating T cells encounter foreign antigen they form long-lived static conjugates with the antigen-presenting cell (APC), while at other times T cells detect foreign antigen but continue to migrate nonetheless. Understanding the determinants of these distinct phenotypes and their immunological consequences is critical for understanding the basic principles of the in vivo immune response. We have previously shown that some chemokines deliver dominant signals that directed T cells to continue migrating past antigen-bearing APCs, while other chemokines deliver subordinant signals that permit long-term conjugate formation. 2 such chemokines are SLC (CCL21, dominant) and SDF-1a (CXCL12, subordinate), recognized by their respective receptors on T cells, CCR7 and CXCR4. Here we propose to design chimeric receptors bearing the ligand-specificity 1 of a dominant receptor and the intracellular signaling response of a subordinate receptor. Because chemokine receptors are 7-transmembrane G-protein coupled receptors, their extracellular, transmembrane and intracellular structures are tightly coupled. In order to discover chimeras with functional ligand binding and appropriate intracellular signaling properties, we propose to generate a combinatorial library of receptor chimeras where all extracellular loops belong to 1 receptor, all intracellular loops belong to the other, and the transmembrane domains are varied between the 2. In Aim 1 we will design and produce the transmembrane-shuffled CCR7/CXCR4 and CCR7/CCR5 libraries by a gene-synthesis approach. In Aims 2 & 3 we will screen this library for desired chemotactic dominant/subordinate behavior. In Aim 4 we will use this data to derive general principles for chemokine receptor transmembrane signaling and test these principles by rational design of a novel chimera between CXCR3 and CCR5. By elucidating the hierarchies of immunological "Stop" and "Go" signals we will reveal basic principles governing immune function that will be relevant to understand how tumors, viruses, and other microbes can sometimes evade immune clearance, and how immunity becomes dysregulated in autoimmune diseases.

描述（由申请人提供）：趋化因子对于引导免疫细胞的运输和组织至关重要。我们和其他人已经证明 T 细胞在淋巴器官内高速迁移。在某些条件下，当迁移的 T 细胞遇到外来抗原时，它们会与抗原呈递细胞 (APC) 形成长期静态结合物，而在其他时候，T 细胞会检测到外来抗原，但仍会继续迁移。了解这些不同表型的决定因素及其免疫学后果对于了解体内免疫反应的基本原理至关重要。我们之前已经证明，一些趋化因子传递主导信号，指导 T 细胞继续迁移经过携带抗原的 APC，而其他趋化因子传递次要信号，允许长期缀合物形成。 2 个这样的趋化因子是 SLC（CCL21，主要）和 SDF-1a（CXCL12，次要），被 T 细胞上各自的受体 CCR7 和 CXCR4 识别。在这里，我们建议设计具有主导受体的配体特异性1和从属受体的细胞内信号传导响应的嵌合受体。由于趋化因子受体是7次跨膜G蛋白偶联受体，因此它们的胞外、跨膜和胞内结构紧密耦合。为了发现具有功能性配体结合和适当的细胞内信号传导特性的嵌合体，我们建议生成受体嵌合体的组合文库，其中所有胞外环属于一个受体，所有胞内环属于另一个受体，并且跨膜结构域在受体嵌合体之间变化。 2. 在目标 1 中，我们将通过基因合成方法设计和生产跨膜改组的 CCR7/CXCR4 和 CCR7/CCR5 文库。在目标 2 和 3 中，我们将筛选该库以获得所需的趋化主导/从属行为。在目标 4 中，我们将使用这些数据推导出趋化因子受体跨膜信号转导的一般原理，并通过合理设计 CXCR3 和 CCR5 之间的新型嵌合体来测试这些原理。通过阐明免疫“停止”和“前进”信号的层次结构，我们将揭示控制免疫功能的基本原理，这些原理将有助于理解肿瘤、病毒和其他微生物有时如何逃避免疫清除，以及免疫在自身免疫性疾病中如何失调。