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

项目摘要 趋化因子控制着白细胞的迁移和定位，在调节白细胞的增殖和分化中起着重要作用。 免疫和炎症反应。其中一种趋化因子CXCL 12促进了肿瘤细胞生长的多个步骤， 许多原发性肿瘤和通过与C-X-C趋化因子受体4（CXCR 4）结合而进展为转移 和非典型趋化因子受体3（ACKR 3），其在许多癌症中上调。与大多数G蛋白不同- 偶联受体（GPCR），CXCL 12结合的ACKR 3仅通过抑制蛋白发出信号。针对CXCL 12， ACKR 3被细胞内GPCR激酶（GRK）磷酸化，随后招募抑制蛋白。的 抑制蛋白用作促进对细胞存活、增殖和迁移至关重要的生长途径的支架。 抑制蛋白还驱动受体内化，在此期间CXCL 12被运输到溶酶体并降解。 然后，空受体再循环到细胞表面，在那里它保持相对稳定的细胞周期。 浓度.该过程导致CXCL 12从细胞外空间的“清除”或摄取， 在正常生理环境中，对于维持CXCR 4表达细胞的反应性是重要的 以及肿瘤转移。在这项提案中，Tesmer和Handel实验室在GRK方面拥有深厚的专业知识， 趋化因子受体的结构和功能，分别加入力量，以更好地了解分子 不同GRK的ACKR 3磷酸化的潜在机制，抑制蛋白如何与产生的 磷酸化“条形码”安装在受体的C末端，以及细胞的后果是什么？ 是.他们发现GRK 2和GRK 5磷酸化激活ACKR 3的不同区域， 胞质尾此外，磷酸化增强与arrestin 2和3的结合。arrestin 2募集 以前没有报道，因此其功能意义仍有待阐明。它们进一步 分离的ACKR 3与抑制蛋白以及与GRK 2-G抑制蛋白的复合物，其具有适合高质量的 分辨率冷冻电子显微镜（cryo-EM）重建，并已表明G蛋白亚基单独可以 与功能未知的ACKR 3形成强相互作用。在Aim 1中，CXCL 12激活的细胞的冷冻-EM结构 ACKR 3将与各种GRK一起确定，重点是GRK 2和G RK。在目标2中，低温- EM将用于检查具有磷酸化ACKR 3的抑制蛋白复合物。在目标3中，假设驱动的细胞- 基于ACKR 3功能的测定和无偏质谱法将用于系统地 研究这些蛋白质控制arrestin介导的信号传导和清除的机制， 并确定是否有特定的GRK和抑制蛋白亚型控制ACKR 3功能。成功 这一建议的结论将导致非典型趋化因子受体的第一个结构， 细胞内信号的合作伙伴，以及前所未有的见解的分子机制， 这些受体可能最终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.