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

项目摘要 CXCL12对趋化因子受体CXCR4和ACKR3的刺激在 发育、免疫系统流动性和炎症反应。这三种基因在癌症中起着重要作用 促进转移和肿瘤增殖。因此，这两种受体都是很有希望的靶点。 治疗，针对CXCR4和ACKR3配体的化合物正在进行临床试验，正在开发中。 尽管有共同的激动剂，CXCR4和ACKR3的反应却截然不同。而CXCR4信号 与其他经典的G蛋白偶联受体(GPCRs)一样，通过G蛋白和β-arrestins，仅ACKR3 被逮捕的夫妇。。CXCR4和ACKR3在结构上相似，并且以相似的方式结合CXCL12 受体似乎通过不同的机制激活。CXCR4对 趋化因子相互作用，单点突变导致高亲和力激动剂作为拮抗剂。 相反，ACKR3是混杂的，几乎所有被测试的配体都起到激动剂的作用。这些差异是如何 转化为有偏见的信号是一个悬而未决的问题。我建议调查CXCL12信号是如何 由这些受体解释，通过解析配体如何影响受体构象和次级 与激酶和β抑制蛋白的相互作用。这一提议的中心假设是，这些相互作用将 对于经典受体和非典型受体是不同的，目前的实验结果将提供 对受体水平的偏向信号的洞察。这一假设将被三个具体目标所追求。目标1： 通过测定高密度脂蛋白来鉴定CXCL12结合后ACKR3的结构重排 拆分该络合物的晶体结构。由此产生的结构将通过以下方式揭示特定的相互作用 CXCL12结合诱导的非典型受体及其激活的结构基础。目标2：确定 CXCL12激活的ACKR3如何被磷酸化并与β-arrestin相互作用。这个目标将决定什么 激酶使ACKR3磷酸化，这些磷酸盐是在哪里结合的，以及 修饰改变β-arrestin的相互作用和信号转导。目标3：确定β-arrestin如何与 CXCL12通过对复合体进行电子显微镜成像来刺激CXCR4，并最终分解 CXCl12：CXCR4：β-Arrestin络合物的结构。总而言之，这些研究将呈现一个无与伦比的观点 受体介导的偏向激动症是如何通过经典和非典型的GPCRs表现出来的。确定如何 这些受体的反应是自然刺激，最终将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.