Biasing CXCR3 Signaling to Modulate the Inflammatory Response

摘要 CXCR3是一种趋化因子受体(CKR)，通过调节T细胞在炎症中发挥重要作用 迁移和功能。尽管CKRs在疾病中的临床相关性已经确立，但只有三个FDA 已批准的针对整个趋化因子系统的药物，该系统由大约20个受体和 50种配体，几乎控制着炎症的方方面面。CKR药物开发困难的原因- 包括给定CKR的多个同源趋化因子配体之间的潜在冗余和缺失 了解由CKRs激活的信号通路如何调节免疫细胞功能和在 火化。因此，针对趋化因子系统的药物存在严重的未得到满足的需求。这让人联想到上下文 我的团队研究趋化因子受体CXCR3。我们已经证明了CXCR3的同源配体， CXCL9、10和11充当有偏向的激动剂，从数量和质量上产生不同的信号 另一种是通过它们与异三聚体G蛋白和β-arrestin接头蛋白的相互作用。在上一次 在项目期间，我们已经发现了小分子G蛋白和β-arrestin偏向的CXCR3激动剂，它们(1)不同于 剧烈激活CXCR3下游的信号通路，以及(2)在小鼠模型中有明显的作用 T细胞介导的炎症性皮肤病。这些发现表明G蛋白和β-arrestin在 促进CXCR3介导的炎症反应。我们研究的长期目标是确定 有偏见的激动症的潜在机制，以开发针对炎症中的CKRs的新疗法。整体而言 这项建议的目的是确定偏向激动剂如何促进不同的效应器构象，从而导致 导致T细胞功能改变和炎症的不同信号模式。我们的中心假设 偏向β-arrestin的激动剂诱导独特的受体和β-arrestin构象有利于“位置双- 促进T细胞转录反应，从而导致不同模式的 发炎。为了达到我们的目标，首先，我们将确定受体：配体复合体如何促进偏向 通过变构调节效应器的反应。我们发现，偏向CXCR3的激动剂促进不同的- Entβ-arrestin介导的作用，我们将使用受体突变体进一步探索这一作用。然后，我们将确定 内体信号和位置偏向如何影响CXCR3偏向信号转导。我们发现有些人 偏向CXCR3的激动剂促进转录，这可以通过抑制受体内吞来阻止。最后， 我们将确定CXCR3G蛋白和β-arrestin介导的信号通路如何影响T细胞功能 以及炎症反应。我们将使用CXCR3突变体来测试特定信号通路的贡献- T细胞体外趋化和体内炎症的途径。这个项目探索了一种创新的研究方法 CXCR3信号，将提供对CXCR3调节炎症反应的了解 选择性激活G蛋白和β阻滞素。这项研究具有重要意义，因为它将为 未来对偏向激动剂作为CXCR3治疗药物的研究，并作为靶向其他CKR的模型。

ABSTRACT CXCR3 is a chemokine receptor (CKR) that plays a central role in inflammation through its regulation of T cell migration and function. Despite the established clinical relevance of CKRs in disease, there are only three FDA approved drugs that target the entire chemokine system, which consists of approximately twenty receptors and fifty ligands that regulate nearly every aspect of inflammation. Reasons for this difficulty in CKR drug develop- ment include the potential redundancy between multiple cognate chemokine ligands for a given CKR and a lack of knowledge regarding how the signaling pathways activated by CKRs regulate immune cell function and in- flammation. Thus, there is a critical unmet need for drugs targeting the chemokine system. This puts into context work from my group on the chemokine receptor CXCR3. We have shown that the cognate ligands of CXCR3, CXCL9, 10 and 11, act as biased agonists, generating quantitatively and qualitatively distinct signals from one another through their interactions with heterotrimeric G proteins and β-arrestin adapter proteins. In the previous project period, we have identified small-molecule G protein- and β-arrestin-biased CXCR3 agonists that (1) dif- ferentially activate signaling pathways downstream of CXCR3, and (2) have distinct effects in a mouse model of T-cell-mediated inflammatory skin disease. These findings suggest distinct roles for G proteins and β-arrestin in promoting the CXCR3-mediated inflammatory response. The long-term goal of our research is to determine the mechanisms underlying biased agonism to develop novel therapies targeting CKRs in inflammation. The overall objective of this proposal is to determine how biased agonists promote different effector conformations that lead to distinct patterns of signaling resulting in changes in T cell function and inflammation. Our central hypothesis is that β-arrestin-biased agonists induce unique receptor and β-arrestin conformations that favor “location bi- ased” endosomal signaling that promotes a T cell transcriptional response that results in different patterns of inflammation. To address our objective, first, we will determine how the receptor:ligand complex promotes biased responses through allosteric regulation of effectors. We have found that CXCR3 biased agonists promote differ- ent β-arrestin-mediated effects, which we will explore further by using receptor mutants. Then, we will determine how endosomal signaling and location bias contribute to CXCR3 biased signaling. We have found that some CXCR3 biased agonists promote transcription that can be prevented by inhibiting receptor endocytosis. Lastly, we will determine how CXCR3 G protein- and β-arrestin-mediated signaling pathways contribute to T cell function and the inflammatory response. We will use CXCR3 mutants to test the contributions of specific signaling path- ways to T cell chemotaxis in vitro and inflammation in vivo. This project explores an innovative approach to study CXCR3 signaling that will provide an understanding of CXCR3 regulation of the inflammatory response by the selective activation of G proteins and β-arrestins. The research is significant as it will lay the groundwork for future research on biased agonists as CXCR3 therapeutics and serve as a model for targeting other CKRs.