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

趋化因子CXCL 12及其G蛋白偶联受体（GPCR）CXCR 4在细胞迁移过程中调节细胞迁移。 发育、免疫监视和正常生理中的炎症。他们也因其 在疾病中的作用，特别是癌症。最近，“非典型”趋化因子受体，ACKR 3，被确定为一种非典型的趋化因子受体。 CXCL 12的第二受体，不通过G蛋白发出信号，而是与β-抑制蛋白偶联。像 CXCR 4、ACKR 3在发育过程中表达，并在癌症中上调。尽管他们的医疗 重要的是，CXCR 4和ACKR 3被激活以引起不同功能反应的机制 我们对此知之甚少。生物物理、计算和诱变研究表明，CXCR 4和 ACKR 3以结构上类似的方式识别CXCL 12。然而，CXCR 4的激活对甚至 趋化因子和受体结合口袋的单点突变，而几乎所有测试的配体 激活ACKR 3。因此，CXCR 4和ACKR 3似乎通过不同的机制起作用。我们假设 CXCR 4活化涉及稳定受体活性构象的精确残基网络， 而ACKR 3激活通过“楔形”机制发生，使得每当任何配体停靠在细胞中时， 受体结合口袋，它激活不稳定的非活性构象。我们建议使用单一- 分子荧光（SMF）光谱，以探索构象动力学和不同的激活 这两种受体的机制。我们还将研究配体和效应物（G蛋白和β- 抑制蛋白）控制CXCR 4和ACKR 3的构象，从而控制它们的信号传导应答。底层 假设是GPCR和ACR本质上是动态的，对多种构象进行采样， 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。我们的中心假设将与三个具体的追求 目标。1：建立SMF方法以实时监测CXCR 4和ACKR 3的构象动力学， 并探索它们的激活机制。2：研究ACKR 3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CXCR 4的结构机制 激动剂结合和G蛋白偶联之间的变构。本提案的创新之处在于 新型SMF方法将提供无法提供的有关受体动力学和变构的实验信息 用其他方法获得。此外，这些方法从未应用于趋化因子 受体之间的关系，很少有人知道构象动力学和非典型 受体激活这些研究意义重大，因为它们将为不同的 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， 一个是β-抑制蛋白偶联的。了解配体和效应子如何控制构象状态， 这些受体的信号输出应最终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.