Some chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits β-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration. Base on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying β-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student’s t-tests or ANOVA using PRISM5 software were employed for statistical analyses. Ligand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via βγ subunits and receptor phosphorylation with subsequent β-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited β-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7. This study demonstrates that SDF-1α-stimulated CXCR7 mediates β-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.

中文翻译：

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CXCR7：β-arrestin偏向受体，可增强细胞迁移并仅通过Gβγ亚基和GRK2募集β-arrestin2。

一些趋化因子受体（非典型趋化因子受体（ACKR））被认为是非信号诱饵，因为它们无法激活典型的G蛋白信号传导途径。CXCR7，也称为ACKR3，仅与两个趋化因子SDF-1α和I-TAC结合，并募集β-arrestin。SDF-1α还与自己的常规受体CXCR4结合，参与体内稳态调节，例如发育和免疫监视以及病理状况，例如炎症，局部缺血和癌症。最近，在这种情况下，CXCR7与CXCR4一起被建议作为关键治疗靶标。然而，尽管进行了大量研究，但仍未阐明细胞应答以及与CXCR7和CXCR4的功能相关的分子机制。因此，我们旨在揭示CXCR7和CXCR4的分子网络，并比较它们对细胞迁移的影响。基于使用NanoBiT技术的结构互补分析，我们表征了CXCR4和CXCR7募集β-arrestin2的不同机制。进行了交联和免疫沉淀以分析受体的复杂形成。使用CRISPR的基因删除和受体的重构被应用于分析依赖配体的ERK磷酸化和细胞迁移。所有实验一式三份进行，并重复三次以上。使用未配对的学生t检验或使用PRISM5软件的ANOVA进行统计分析。配体与CXCR7的结合不会导致通过Gα亚基激活典型的信号通路，但会导致通过βγ亚基激活GRK2和受体磷酸化以及随后的β-arrestin2募集。相反，CXCR4诱导Gαi活化并通过GRK2和GRK5的C末端磷酸化募集β-arrestin2。CXCR4促进了SDF-1α刺激的ERK磷酸化，但CXCR7却没有。这项研究中没有证实CXCR4和CXCR7的异二聚化，而通过交联实验和NanoBiT分析证实了它们的均二聚化。关于趋化性，SDF-1α刺激的细胞迁移是由CXCR4和CXCR7介导的。这项研究表明，SDF-1α刺激的CXCR7通过与CXCR4不同的分子网络介导β-arrestin2募集。