Amsterdam Institute for Molecular and Life Sciences (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Institut de Génomique Fonctionnelle (IGF), Université de Montpellier, CNRS, INSERM, 34094 Montpellier, France.
Cells. 2021 Mar 11;10(3):618. doi: 10.3390/cells10030618.
: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. : We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. : Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T and in part S are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.
: 非典型趋化因子受体 3（ACKR3）属于 G 蛋白偶联受体（GPCR）超家族。与经典 GPCR 不同，该受体在大多数细胞类型中不激活 G 蛋白，但在激活时募集β-arrestin。ACKR3 在癌症和血管疾病中发挥重要作用。由于 GPCR 羧基末端尾巴的磷酸化触发β-arrestin 的募集，我们研究了 ACKR3 C 尾巴中不同潜在磷酸化位点的作用，以进一步描绘这种 GPCR 内化和运输的分子机制。: 我们使用各种生物发光和荧光共振能量转移（FRET）基于传感器和技术在表达 WT 或磷酸化位点突变体的人胚肾（HEK）293T 细胞中测量 CXCL12 诱导的β-arrestin 和 G 蛋白偶联受体激酶（GRK）募集、受体内化和运输。: 在 CXCL12 刺激下，ACKR3 以相同的动力学特征募集β-arrestin1 和β-arrestin2。我们确定了与 GRK2、3 和 5 的相互作用，GRK2 和 3 对β-arrestin 募集很重要。激活后，ACKR3 内化并重新循环到细胞膜。我们证明，受体上β-arrestin 的募集主要由 ACKR3 C 尾上一个磷酸化残基簇决定，并且残基 T 和部分 S 是β-arrestin1 募集的重要残基。C 尾的磷酸化似乎对内化诱导和差异β-arrestin 募集很重要。GRK2 和 3 在受体内化中起关键作用。此外，当β-arrestin 募集受损或不存在β-arrestin 时，ACKR3 仍然可以使用替代内化途径内化。我们的数据表明，ACKR3 C 尾中的不同残基差异调节 CXCL12 诱导的β-arrestin 募集、ACKR3 运输和内化。
