The chemokine receptor CXCR4 plays a fundamental role in homeostasis and pathology by orchestrating recruitment and positioning of immune cells, under the guidance of a CXCL12 gradient. The ability of chemokines to form heterocomplexes, enhancing their function, represents an additional level of regulation on their cognate receptors. In particular, the multi-faceted activity of the heterocomplex formed between CXCL12 and the alarmin HMGB1 is emerging as an unexpected player able to modulate a variety of cell responses, spanning from tissue regeneration to chronic inflammation. Nowadays, little is known on the selective signaling pathways activated when CXCR4 is triggered by the CXCL12/HMGB1 heterocomplex. In the present work, we demonstrate that this heterocomplex acts as a CXCR4 balanced agonist, activating both G protein and β-arrestins-mediated signaling pathways to sustain chemotaxis. We generated β-arrestins knock out HeLa cells by CRISPR/Cas9 technology and show that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization is primarily β-arrestin1 dependent, while chemotaxis requires both β-arrestin1 and β-arrestin2. Triggering of CXCR4 with the CXCL12/HMGB1 heterocomplex leads to an unexpected receptor retention on the cell surface, which depends on β-arrestin2. In conclusion, the CXCL12/HMGB1 heterocomplex engages the β-arrestin proteins differently from CXCL12, promoting a prompt availability of CXCR4 on the cell surface, and enhancing directional cell migration. These data unveil the signaling induced by the CXCL12/HMGB1 heterocomplex in view of identifying biased CXCR4 antagonists or agonists targeting the variety of functions it exerts.

趋化因子受体CXCR4在CXCL12梯度的指导下，通过协调免疫细胞的募集和定位，在体内稳态和病理中起着基本作用。趋化因子形成异源复合物，增强其功能的能力代表了对其同源受体的额外调节。尤其是，CXCL12和警报蛋白HMGB1之间形成的异源复合物的多方面活性正成为意料之外的参与者，能够调节从组织再生到慢性炎症的各种细胞反应。如今，关于由CXCL12 / HMGB1异源复合物触发CXCR4时激活的选择性信号通路的了解甚少。在目前的工作中，我们证明了这种杂合物可作为CXCR4平衡激动剂，激活G蛋白和β-arrestins介导的信号通路以维持趋化性。我们通过CRISPR / Cas9技术生成了β-arrestin敲除HeLa细胞，并显示CXCL12 / HMGB1异源复合物介导的肌动蛋白聚合主要依赖β-arrestin1，而趋化性同时需要β-arrestin1和β-arrestin2。用CXCL12 / HMGB1异源复合物触发CXCR4会导致意外的受体保留在细胞表面，这取决于β-arrestin2。总之，CXCL12 / HMGB1异源复合物与CXCL12的结合方式不同，其参与β-arrestin蛋白的结合，从而促进了CXCR4在细胞表面的迅速利用，并增强了细胞的定向迁移。