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

趋化因子CXCL12及其G蛋白偶联受体CXCR4调节细胞迁移。 正常生理中的发育、免疫监测和炎症。他们还因为他们的 在疾病中的作用，尤其是癌症。最近，非典型的趋化因子受体ACKR3被鉴定为一种 CXCL12的第二个受体，不通过G蛋白传递信号，而是与β-arrestin偶联。喜欢 CXCR4、ACKR3在发育过程中表达，在肿瘤中表达上调。尽管他们的体检 重要性，CXCR4和ACKR3被激活以引起不同功能反应的机制 人们对此了解甚少。生物物理、计算和诱变研究表明，CXCR4和 ACKR3以一种结构相似的方式识别CXCL12。然而，CXCR4的激活对EVEN敏感 趋化因子和受体结合口袋的单点突变，而几乎所有的配体都进行了测试 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的激活涉及一个精确的残基网络，稳定受体的活性构象， 鉴于ACKR3的激活是通过一种“楔形”机制进行的，因此每当有任何配体停靠在 受体结合口袋，它通过破坏非活性构象的稳定而激活。我们建议使用单人- 分子荧光光谱研究构象动力学和不同活化度 这两种受体的作用机制。我们还将研究配体和效应器(G蛋白和β- Arrestin)控制CXCR4和ACKR3的构象，从而控制它们的信号反应。潜在的 假设GPCR和ACR本质上是动态的，采样多个构象，并且 配体和效应物相互调节，影响受体的构象和信号输出。 强劲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， 并探讨其激活机制。2：研究ACKR3激活的结构机制和 激动剂结合和β-arrestin偶联之间的变构。3：研究CXCR4的结构机制 激动剂结合与G蛋白偶联之间的激活和变构。这一建议的创新之处在于 这种新的SMF方法将提供关于受体动力学和变构的实验信息，而不是 可以用其他方法获得。此外，这些方法从未应用于趋化因子。 受体，对构象动力学和非典型之间的关系知之甚少 受体激活。这些研究具有重要意义，因为它们将为不同的 两个具有重要治疗意义的受体的激活机制，一个是G蛋白偶联受体，另一个是 一种是β-arrestin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会为药物开发提供信息。

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.