Insights into Activation Mechanisms of G Protein-Coupled and Atypical β-Arrestin-Coupled Chemokine Receptors

深入了解 G 蛋白偶联和非典型 β-抑制蛋白偶联趋化因子受体的激活机制

• 批准号: 9899267
• 负责人: Tracy M Handel
• 金额: $ 45.2万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899267
• 关键词: Agonist; Arrestins; Binding; Binding Proteins; Biophysics; CXCL12 gene; CXCR4 gene; Cardiovascular system; Clinical Trials; Coupled; Couples; Coupling; Data; Development; Disease; Docking; Drug Targeting; Fluorescence; Fluorescence Spectroscopy; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Immunologic Surveillance; Immunosuppression; Impairment; Inflammation; Inflammatory; Ligand Binding; Ligands; Malignant Neoplasms; Medical; Methods; Modeling; Molecular Conformation; Monitor; Mutagenesis; Mutation; Neoplasm Metastasis; Output; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiology; Point Mutation; Property; Proteins; Public Health; Receptor Activation; Research; Role; Sampling; Signal Transduction; Structural Models; Structure; Testing; Therapeutic; Time; angiogenesis; arrestin 2; beta-arrestin; cancer therapy; cell motility; chemokine; chemokine receptor; conformational conversion; drug action; drug development; improved; inhibitor/antagonist; innovation; insight; novel; receptor; receptor binding; receptor function; response; side effect; single molecule; small molecule; tumor growth; virtual

The chemokine CXCL12 and its G protein-coupled receptor (GPCR), CXCR4, regulate cell migration during development, immune surveillance and inflammation in normal physiology. They are also notorious for their roles in disease, particularly cancer. Recently, the "atypical" chemokine receptor, ACKR3, was identified as a second receptor for CXCL12 that does not signal through G proteins but instead couples to β-arrestin. Like CXCR4, ACKR3 is expressed during development and up-regulated in cancer. Despite their medical importance, the mechanisms by which CXCR4 and ACKR3 are activated to elicit distinct functional responses are poorly understood. Biophysical, computational and mutagenesis studies have shown that CXCR4 and ACKR3 recognize CXCL12 in a structurally similar manner. However, activation of CXCR4 is sensitive to even single point mutations of the chemokine and the receptor-binding pocket, whereas virtually all ligands tested activate ACKR3. Thus, CXCR4 and ACKR3 appear to function by different mechanisms. We hypothesize that CXCR4 activation involves a precise network of residues that stabilize the active conformation of the receptor, whereas ACKR3 activation occurs by a “wedge-like” mechanism, such that whenever any ligand docks in the receptor-binding pocket, it activates by destabilizing the inactive conformation. We propose to use single- molecule fluorescence (SMF) spectroscopy to explore the conformational dynamics and different activation mechanisms of these two receptors. We will also investigate how ligands and effectors (G proteins and β- arrestin) control the conformations of CXCR4 and ACKR3 and thus their signaling responses. The underlying hypothesis is that GPCRs and ACRs are intrinsically dynamic, sampling multiple conformations, and that ligands and effectors mutually regulate each other to influence the receptor conformation and signaling output. Strong preliminary data support this hypothesis. Our central hypothesis will be pursued with three specific aims. 1: Establish SMF methods to monitor the conformational dynamics of CXCR4 and ACKR3 in real-time, and probe their mechanisms of activation. 2: Investigate structural mechanisms of ACKR3 activation and the allostery between agonist binding and β-arrestin coupling. 3: Investigate structural mechanisms of CXCR4 activation and the allostery between agonist binding and G protein coupling. The innovation of this proposal is that novel SMF methods will provide experimental information on receptor dynamics and allostery that cannot be obtained with other methods. Moreover, these approaches have never been applied to chemokine receptors and very little is known about the relationship between conformational dynamics and atypical receptor activation. The studies are significant because they will provide unique insights into the distinct activation mechanisms of two therapeutically important receptors, one that is a G protein-coupled receptor and one that is β-arrestin-coupled. Understanding how ligands and effectors control the conformational state and signaling output of these receptors should ultimately inform drug development.

趋化因子CXCL12及其G蛋白偶联受体（GPCR）CXCR4调节细胞迁移。 正常生理学中的发育，免疫监测和感染。他们也因他们的 在疾病中的作用，尤其是癌症。最近，“非典型”趋化因子受体ACKR3被确定为A CXCL12的第二个受体不会通过G蛋白发出信号，而是伴侣与β-arrestin伴侣。喜欢 CXCR4，ACKR3在发育过程中表达并在癌症中上调。尽管有医疗 重要性，激活CXCR4和ACKR3的机制以引起不同的功能响应 知之甚少。生物物理，计算和诱变研究表明，CXCR4和 ACKR3以结构相似的方式识别CXCL12。但是，CXCR4的激活对甚至 趋化因子和接收器结合口袋的单点突变，而几乎所有测试的配体 激活ACKR3。这，CXCR4和ACKR3似乎通过不同的机制起作用。我们假设这一点 CXCR4激活涉及一个精确的残差网络，可稳定接收器的主动构象， 而ACKR3激活是通过“楔形”机制发生的，以便每当任何配体在码头中 受体结合口袋，它通过破坏无效构象而激活。我们建议使用单人 分子荧光（SMF）光谱学探索构象动力学和不同的激活 这两个受体的机制。我们还将研究配体和作用如何（G蛋白和β- 逮捕素）控制CXCR4和ACKR3的构象，从而信号反应。基础 假设是GPCR和ACR是本质上动态的，对多种构象进行了采样，并且 配体和效应相互调节，以影响受体构象和信号输出。 强大的初步数据支持这一假设。我们的中心假设将被三个特定的 目标。 1：建立SMF方法以实时监视CXCR4和ACKR3的构象动力学， 并探测它们的激活机制。 2：研究ACKR3激活的结构机制和 激动剂结合和β-甲蛋白耦合之间的变构。 3：研究CXCR4的结构机制 激活和古老结合与G蛋白偶联之间的变构。该提议的创新是 这种新颖的SMF方法将提供有关受体动力学和变构的实验信息 可以使用其他方法获得。此外，这些方法从未应用于趋化因子 受体和对构象动力学与非典型的关系知之甚少 受体激活。研究之所以重要，是因为它们将为独特的独特见解提供独特的见解 两种具有治疗疗法的受体的激活机制，一种是G蛋白偶联受体和 一种是β-arrestin耦合。了解配体和作用如何控制构象状态和 这些受体的信号传导输出最终应为药物开发提供信息。