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 在细胞迁移过程中调节细胞迁移 正常生理学中的发育、免疫监视和炎症。他们也因自己的行为而臭名昭著 在疾病，特别是癌症中的作用。最近，“非典型”趋化因子受体 ACKR3 被确定为 CXCL12 的第二个受体不通过 G 蛋白发出信号，而是与 β-arrestin 偶联。喜欢 CXCR4、ACKR3 在发育过程中表达，并在癌症中上调。尽管他们的医疗 重要性，CXCR4 和 ACKR3 被激活以引发不同功能反应的机制 人们了解甚少。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。然而，CXCR4 的激活甚至对 趋化因子和受体结合袋的单点突变，而几乎所有测试的配体 激活ACKR3。因此，CXCR4 和 ACKR3 似乎通过不同的机制发挥作用。我们假设 CXCR4 激活涉及稳定受体活性构象的精确残基网络， 而 ACKR3 激活是通过“楔形”机制发生的，这样每当任何配体停靠在 受体结合口袋，它通过破坏非活性构象的稳定性来激活。我们建议使用单 分子荧光（SMF）光谱探索构象动力学和不同的激活 这两种受体的作用机制。我们还将研究配体和效应子（G 蛋白和 β- 抑制蛋白）控制 CXCR4 和 ACKR3 的构象，从而控制它们的信号反应。底层的 假设 GPCR 和 ACR 本质上是动态的，采样多种构象，并且 配体和效应子相互调节，影响受体构象和信号输出。 强有力的初步数据支持这一假设。我们的中心假设将通过三个具体的 目标。 1：建立SMF方法实时监测CXCR4和ACKR3的构象动力学， 并探究其激活机制。 2：研究 ACKR3 激活的结构机制和 激动剂结合和β-抑制蛋白偶联之间的变构。 3：研究CXCR4的结构机制 激动剂结合和 G 蛋白偶联之间的激活和变构。本提案的创新点在于 新颖的 SMF 方法将提供有关受体动力学和变构的实验信息，而这是无法提供的 可以通过其他方法获得。此外，这些方法从未应用于趋化因子 受体，但对于构​​象动力学和非典型之间的关系知之甚少 受体激活。这些研究意义重大，因为它们将为不同的领域提供独特的见解。 两种治疗上重要的受体的激活机制，一种是 G 蛋白偶联受体， 一种是β-抑制蛋白偶联的。了解配体和效应子如何控制构象状态和 这些受体的信号输出最终应该为药物开发提供信息。