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

GPCR 激酶、Gβγ 和抑制蛋白对促进趋化因子受体 ACKR3 的转移的调节

基本信息

批准号：
10397636
负责人：
Tracy M Handel
金额：
$ 63.18万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10397636
关键词：
ADRBK1 gene Arrestins Bar Codes Binding Biological Assay CXCL12 gene CXCR4 Receptors Cell Surface Receptors Cell Survival Cell membrane Cell surface Cells Clinical Trials Complex Couples Critical Pathways Cryoelectron Microscopy Cytoplasmic Tail Data Development Disease Endothelial Cells Extracellular Space G protein coupled receptor kinase G-Protein-Coupled Receptors GRK5 gene GTP-Binding Proteins Goals Growth Hand Heterotrimeric GTP-Binding Proteins Immune response Inflammatory Response Lead Leukocytes Ligands Lysosomes Malignant Neoplasms Mass Spectrum Analysis Mediating Metastatic to Molecular Molecular Conformation Neoplasm Metastasis Outcome Pathway interactions Phenotype Phosphorylation Phosphotransferases Physiology Play Primary Neoplasm Process Protein Isoforms Proteins Proto-Oncogene Proteins c-akt Regulation Reporting Research Resolution Role Side Signal Transduction Site Solid Neoplasm Structure Therapeutic arrestin3 base biophysical techniques cancer cell cancer therapy cell motility chemokine chemokine receptor desensitization extracellular insight leukemia/lymphoma migration receptor receptor internalization receptor structure function reconstruction recruit response scaffold structural biology tumor tumor growth tumor progression uptake

项目摘要

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.

项目摘要趋化因子控制白细胞的迁移和定位，并在调节中起基本作用免疫和炎症反应。一种这样的趋化因子CXCL12，促进了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型（CXCR4）结合而发展为转移和非典型趋化因子受体3（ACKR3），在许多癌症中都进行了更新。与大多数G蛋白不同耦合受体（GPCR），仅通过逮捕蛋白信号CXCL12结合的ACKR3信号。响应CXCL12， ACKR3被细胞内GPCR激酶（GRKS）磷酸化，随后募集了抑制蛋白。这逮捕蛋白作为脚手架，促进对细胞存活，增殖和迁移至关重要的生长途径。逮捕蛋白还驱动受体内在化，在此期间，CXCL12被贩运到溶酶体并降解。之后，将空接收器回收到细胞表面，并保持相对稳定专注。此过程导致从细胞外空间和对于在正常生理的背景下维持表达CXCR4细胞的反应性很重要以及肿瘤转移。在此提案中，Tesmer和Handel Labs，在GRK和趋化因子受体的结构和功能分别结合力，以更好地了解分子不同GRK的ACKR3磷酸化的机制，逮捕蛋白如何与结果相互作用接收器的C末端安装的磷酸化“条形码”，以及细胞后果是。他们发现GRK2和GRK5磷酸化在其不同区域激活了ACKR3 细胞质尾巴。此外，磷酸化增强了与逮捕素2和3的结合。以前没有报道过，因此其功能意义尚待阐明。他们有进一步的 ACKR3的孤立复合物具有抑制蛋白以及GRK2 –G的配合分辨率冷冻电子显微镜（Cryo-EM）重建，并表明单独的亚基可以与未知功能的ACKR3形成强烈的相互作用。在AIM1中，CXCL12激活的冷冻EM结构 ACKR3将与各种grks一起确定，重点是GRK2和G。在AIM2中，冷冻 EM将用于检查与磷酸化ACKR3的抑制蛋白复合物。在AIM 3中，假设驱动细胞 - 基于ACKR3功能和无偏质谱法的基于基于的测定将用于系统地研究这些蛋白质控制逮捕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ACKR3功能的抑制素同工型对照。成功该提案的结论将导致与其复合物中非典型趋化因子受体的第一个结构细胞内信号伴侣以及对A分子机制的前所未有的见解在治疗上重要的受体，最终可能有助于开发新的癌症治疗。