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

项目概要 趋化因子控制白细胞的迁移和定位，并在调节中发挥重要作用 免疫和炎症反应。其中一种趋化因子 CXCL12 可促进细胞生长的多个步骤 许多原发性肿瘤通过与 C-X-C 趋化因子受体 4 型 (CXCR4) 结合而进展为转移 和非典型趋化因子受体 3 (ACKR3)，它们在许多癌症中表达上调。与大多数 G 蛋白不同 - 耦合受体 (GPCR) 中，CXCL12 结合的 ACKR3 信号仅通过抑制蛋白发出。响应 CXCL12， ACKR3 被细胞内 GPCR 激酶 (GRK) 磷酸化，随后招募抑制蛋白。的 视紫红质抑制蛋白充当支架，促进对细胞存活、增殖和迁移至关重要的生长途径。 抑制蛋白还驱动受体内化，在此期间 CXCL12 被运输到溶酶体并被降解。 然后，空的受体被回收到细胞表面，在那里保持相对稳定的状态。 浓度。该过程导致 CXCL12 从细胞外空间“清除”或摄取，并 对于在正常生理情况下维持表达 CXCR4 的细胞的反应性非常重要 还有肿瘤的转移。在这项提案中，Tesmer 和 Handel 实验室在 GRK 和 趋化因子受体的结构和功能分别联手更好地了解分子 不同 GRK 磷酸化 ACKR3 的机制，抑制蛋白如何与所得的相互作用 安装在受体 C 末端的磷酸化“条形码”及其细胞后果 是。他们发现GRK2和GRK5在其不同区域磷酸化激活ACKR3。 细胞质尾。此外，磷酸化增强了与抑制蛋白 2 和 3 的结合。抑制蛋白 2 招募 此前未见报道，因此其功能意义仍有待阐明。他们还有进一步 ACKR3 与视紫红质抑制蛋白以及 GRK2–G 的分离复合物，其质量适合高 分辨率冷冻电子显微镜 (cryo-EM) 重建并表明单独的 G 亚基可以 与未知功能的ACKR3形成强相互作用。在 Aim1 中，CXCL12 激活的冷冻电镜结构 ACKR3 将与各种 GRK 综合确定，重点是 GRK2，并与 G 一起确定。在 Aim2 中，冷冻 EM 将用于检查抑制蛋白与磷酸化 ACKR3 的复合物。在目标 3 中，假设驱动的细胞- 基于 ACKR3 功能的测定和无偏质谱方法将用于系统地 研究这些蛋白质控制视紫红质抑制蛋白介导的信号传导和清除的机制 ACKR3并确定是否存在特定的GRK和抑制蛋白亚型控制ACKR3功能。成功者 该提案的结论将导致非典型趋化因子受体与其复合物的第一个结构 细胞内信号传导伙伴以及对细胞内分子机制的前所未有的见解 治疗上重要的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.