Regulation of the metastasis promoting chemokine receptor ACKR3 by GPCR kinases, Gβγ and arrestins

项目总结 趋化因子控制白细胞的迁移和定位，并在调节中发挥基础性作用 免疫和炎症反应。CXCL12就是一种这样的趋化因子，它促进了肿瘤生长的多个步骤 许多原发肿瘤与C-X-C趋化因子受体4(CXCR4)结合而发生转移 和非典型趋化因子受体3(ACKR3)，它们在许多癌症中上调。与大多数G蛋白不同- 偶联受体(GPCRs)，CXCL12结合的ACKR3信号仅通过阻滞素。为响应CXCL12， ACKR3被细胞内GPCRK(GRKs)磷酸化，GRKs随后招募阻滞剂。这个 阻滞素作为支架，促进对细胞存活、增殖和迁移至关重要的生长途径。 抑制素还推动受体内化，在此过程中，CXCL12被运输到溶酶体并被降解。 然后，空受体被循环到细胞表面，在那里它保持相对稳定的 集中精神。这一过程导致CXCL12从细胞外空间中被“清除”或摄取 在正常生理环境下，对维持表达CXCR4的细胞的反应性很重要 以及肿瘤转移。在这项提案中，在GRK和GRK方面拥有深厚专业知识的特斯默和亨德尔实验室 趋化因子受体的结构和功能分别联合起来，更好地理解分子 不同GRK磷酸化ACKR3的机制，拦阻蛋白如何与结果相互作用 安装在受体C-末端的磷酸化“条形码”，以及细胞后果 是。他们发现，GRK2和GRK5磷酸化激活了ACKR3在其不同的区域 细胞质尾巴。此外，磷酸化增强了与arrestin2和3的结合。 尚未见报道，因此其功能意义尚待阐明。他们有更多的 AKR3与Arrestin以及GRK2-G的分离络合物具有较高的质量 分辨率冷冻电子显微镜(CRYO-EM)重建，并表明G亚基单独可以 与未知功能的ACKR3形成强相互作用。在Aim1中，CXCL12激活的低温EM结构 ACKR3将在与各种GRK的络合物中确定，重点是GRK2和G。在AIM2，低温- 电子显微镜将被用于研究arrestin与磷酸化的ACKR3的络合物。在目标3中，假设驱动的细胞- 基于ACKR3功能的分析和无偏质量谱方法将被用于系统地 研究这些蛋白质通过以下途径控制arrestin介导的信号和清除的机制 并确定ACKR3功能是否存在特异的GRK和arrestin异构体控制。成功者 这一提议的结论将导致非典型趋化因子受体的第一个结构与其 细胞内信号转导伙伴，以及对一种 治疗上重要的受体，最终可能有助于开发新的癌症治疗方法。

PROJECT SUMMARY Chemokines control the migration and localization of leukocytes and play fundamental roles in regulating immune and inflammatory responses. One such chemokine, CXCL12, promotes multiple steps in the growth of many primary tumors and progression to metastasis by binding to C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), which are upregulated in many cancers. Unlike most G protein- coupled receptors (GPCRs), CXCL12-bound ACKR3 signals only via arrestins. In response to CXCL12, ACKR3 is phosphorylated by intracellular GPCR kinases (GRKs) which subsequently recruit arrestins. The arrestins serve as scaffolds that promotes growth pathways critical for cell survival, proliferation, and migration. Arrestins also drive receptor internalization, during which CXCL12 is trafficked to lysosomes and degraded. Afterwards, the empty receptor is recycled to the cell surface where it maintains a relatively stable concentration. This process results in the "scavenging" or uptake of CXCL12 from the extracellular space and is important for maintaining the responsiveness of CXCR4-expressing cells in the context of normal physiology as well as tumor metastasis. In this proposal, the Tesmer and Handel labs, with deep expertise in GRKs and chemokine receptor structure and function, respectively, join forces to better understand the molecular mechanisms underlying ACKR3 phosphorylation by different GRKs, how arrestins interact with the resulting phosphorylation “barcodes” installed in the C-terminus of the receptor, and what the cellular consequences are. They have discovered that GRK2 and GRK5 phosphorylate activated ACKR3 at distinct regions of its cytoplasmic tail. Moreover, phosphorylation enhances binding to both arrestin2 and 3. Arrestin2 recruitment has not been reported before, and thus its functional significance remains to be elucidated. They have further isolated complexes of ACKR3 with both arrestin as well as with GRK2–Gthat are of suitable quality for high resolution cryo-electron microscopy (cryo-EM) reconstructions and have shown that G subunits alone can form a strong interaction with ACKR3 of unknown function. In Aim1, cryo-EM structures of CXCL12-activated ACKR3 will be determined in complex with various GRKs, with focus on GRK2, and with G. In Aim2, cryo- EM will be used to examine arrestin complexes with phosphorylated ACKR3. In Aim 3, hypothesis driven cell- based assays of ACKR3 function and unbiased mass spectrometry approaches will be used to systematically investigate mechanisms by which these proteins control arrestin-mediated signaling and scavenging by ACKR3 and determine if there is specific GRK and arrestin isoform control of ACKR3 function. The successful conclusion of this proposal will result in the first structure of an atypical chemokine receptor in complex with its intracellular signaling partners as well as unprecedented insights into the molecular mechanisms of a therapeutically important receptor that may ultimately aid in the development of new cancer treatments.