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CCR6 is a G protein-coupled receptor (GPCR) that recognizes a single chemokine ligand, CCL20 and is primarily expressed by leukocytes. Upon ligand binding, CCR6 activates Gαi heterotrimeric G proteins to induce various potential cellular outcomes through context-specific cell signaling. It is well known that differential phosphorylation of Ser and Thr residues in the C-terminal domains or intracellular loops of GPCRs can generate barcodes that regulate GPCR function by regulating the recruitment of β-arrestins. In this study, we demonstrate that ligand binding to CCR6 induces receptor phosphorylation at Ser/Thr residues in the C-terminal tail, rather than intracellular loops. Using mutagenesis experiments, we determined that distinct clusters of Ser/Thr residues in the C-terminal domain differentially regulate CCL20-induced signaling and cellular response. Substituting the Thr360/Ser361/Thr363 cluster or the Ser370/Ser371 cluster with Ala residues modulated cellular response upon CCL20 stimulation. Notably, receptor internalization, chemotaxis, F-actin distribution, transient ERK1/2 activation, and β-arrestin 2 recruitment were oppositely affected by mutating the two clusters, suggesting that phosphorylation of CCR6 C-terminal Ser/Thr residues directs the cell signaling response upon receptor activation. Moreover, activated CCR6 weakly recruited β-arrestin 1 in comparison with β-arrestin 2, and the two arrestin proteins seemed to play overlapping but distinct roles in mediating CCL20/CCR6-induced cellular responses. Taken together, the effects of site-specific Ser/Thr phosphorylation on CCR6 demonstrate the existence of barcodes on the protein that dictate the activation of different cell signaling profiles and lead to distinct biological outcomes.

CCR6是一种仅识别单一趋化因子配体CCL20的G蛋白偶联受体（G protein-coupled receptor, GPCR），主要由白细胞（leukocytes）表达。配体结合后，CCR6可激活Gαi异源三聚体G蛋白，通过情境特异性细胞信号通路诱导多种潜在的细胞效应。众所周知，G蛋白偶联受体C端结构域或胞内环内的丝氨酸（Ser）与苏氨酸（Thr）残基发生差异化磷酸化，可通过调控β抑制蛋白（β-arrestins）的招募，生成调控受体功能的“条形码”。本研究证实，配体与CCR6结合会诱导受体C端尾区而非胞内环内的Ser/Thr残基发生磷酸化。通过诱变实验，我们明确了C端结构域内不同的Ser/Thr残基簇，可差异化调控CCL20诱导的信号通路与细胞应答。将Thr360/Ser361/Thr363残基簇或Ser370/Ser371残基簇替换为丙氨酸（Ala）残基，会改变CCL20刺激下的细胞应答。值得注意的是，突变这两个残基簇会对受体内吞、趋化性、丝状肌动蛋白（F-actin）分布、瞬时ERK1/2激活以及β抑制蛋白2（β-arrestin 2）招募产生相反的影响，这表明CCR6 C端Ser/Thr残基的磷酸化，可在受体激活后指导细胞信号应答的走向。此外，相较于β抑制蛋白2，激活状态的CCR6仅能微弱招募β抑制蛋白1（β-arrestin 1），且两种抑制蛋白在介导CCL20/CCR6诱导的细胞应答时，呈现功能重叠但作用各异的特点。综上，位点特异性Ser/Thr磷酸化对CCR6的调控作用，证明该蛋白上存在可决定不同细胞信号通路激活模式、并最终导致差异化生物学效应的“条形码”。