Biased agonsim of CXCL12 stimulation of the atypical and classical receptors, ACKR3 and CXCR4

项目摘要 趋化因子受体CXCR 4和ACKR 3的CXCL 12刺激在细胞周期中驱动细胞迁移。 发育、免疫系统移动性和炎症反应。这三个在癌症中起着重要作用 在那里它们促进转移和肿瘤增殖。因此，这两种受体都是有希望的靶点， 目前正在开发针对CXCR 4和ACKR 3配体的化合物的临床试验。 尽管有共同的激动剂，CXCR 4和ACKR 3有明显不同的反应。虽然CXCR 4信号 与其他经典的G蛋白偶联受体（GPCR）一样，通过G蛋白和β-抑制蛋白， 夫妇与逮捕。. CXCR 4和ACKR 3在结构上相似，并且以相似的方式结合CXCL 12，但 受体似乎通过不同的机制激活。CXCR 4对细胞的变化非常敏感， 趋化因子相互作用，单点突变导致高亲和力激动剂充当拮抗剂。 相反，ACKR 3是混杂的，几乎所有测试的配体都充当激动剂。这些差异如何 转化为有偏见的信号是一个悬而未决的问题。我建议研究CXCL 12信号是如何 这些受体通过解析配体如何影响受体构象和二级结构来解释。 与激酶和β-抑制蛋白的相互作用。该建议的核心假设是，这些相互作用将 对于经典和非典型受体是不同的，并且所提出的实验的结果将提供 深入了解受体水平的偏置信号。这一假设将通过三个具体目标来实现。目标1： 通过测定CXCL 12结合诱导的ACKR 3的高水平重排来鉴定CXCL 12结合诱导的ACKR 3的结构重排。 解析配合物的晶体结构。由此产生的结构将揭示特定的相互作用， 一种由CXCL 12结合和激活的结构基础诱导的非典型受体。目标2：确定 CXCL 12激活的ACKR 3如何磷酸化并与β-抑制蛋白相互作用。这一目标将决定什么 激酶磷酸化的ACKR 3，这些磷酸盐被纳入和如何模式， 修饰改变β-抑制蛋白相互作用和信号传导。目的3：确定β-抑制蛋白如何与 CXCL 12刺激的CXCR 4通过电子显微镜对复合物进行成像，并最终解析CXCL 12刺激的CXCR 4。 CXCL 12：CXCR 4：β-抑制蛋白复合物的结构。这些研究将为我们提供一个 受体介导的偏向性激动作用如何通过经典和非典型GPCR表现出来。确定如何 这些受体对自然刺激的反应将最终促进未来的药物开发。

Project Abstract CXCL12 stimulation of the chemokine receptors CXCR4 and ACKR3 drives cellular migration during development, immune system mobility, and inflammatory responses. The trio plays a major role in cancers where they promote metastasis and tumor proliferation. Thus, both receptors are promising targets for therapeutics, with ongoing clinical trials of compounds targeting CXCR4 and ACKR3 ligands in development. Despite the common agonist, CXCR4 and ACKR3 have decidedly different responses. While CXCR4 signals through both G proteins and β-arrestins like other classical G protein-coupled receptors (GPCRs), ACKR3 only couples with arrestins. . CXCR4 and ACKR3 are structurally alike and bind CXCL12 in a similar manner, yet the receptors appear to activate by different mechanisms. CXCR4 is extremely sensitive to changes to the chemokine interaction, with single-point mutations leading the high-affinity agonist to act as an antagonist. Conversely, ACKR3 is promiscuous, with nearly all ligands tested acting as agonists. How these differences translate into the biased signaling is an open question. I propose to investigate the how the CXCL12 signal is interpreted by these receptors by resolving how the ligand affects the receptor conformation and the secondary interactions with kinases and β-arrestins. The central hypothesis of this proposal is that these interactions will be different for the classical and atypical receptors and the results of the presented experiments will provide insights into receptor-level biased signaling. This hypothesis will be pursued by three specific aims. Aim 1: Identify the structural rearrangements of ACKR3 induced by CXCL12 binding by determining the high resolution crystal structure of the complex. The resulting structure will reveal the specific interactions through an atypical receptor that are induced by CXCL12 binding and structural basis of activation. Aim 2: Determine how CXCL12-activated ACKR3 is phosphorylated and interacts with β-arrestin. This aim will determine what kinases phosphorylate the ACKR3, where those phosphates are incorporated and how the pattern of modification alters β-arrestin interactions and signaling. Aim 3: Determine how β-arrestin interacts with CXCL12-stimulated CXCR4 by imaging the complex with electron microscopy and ultimately resolving the structure of the CXCL12:CXCR4:β-arrestin complex. Together, these studies will present an unparalleled view into how receptor-mediated biased agonism is manifested by classical and atypical GPCRs. Determining how these receptors respond the natural stimulus will ultimately facilitate future drug development.