XCR1与CXCR4形成异源二聚体并调控其内化(英文)  

XCR1 Forms a Heterodimer with CXCR4 and Regulates Its Internalization

作　　者：叶鑫 张蕾[2] 成璐[3] 贾岩 任杉 刘冰 宋露瑶 王思懿 李京敬[1] YE Xin;ZHANG Lei;CHENG Lu;JIA Yan;REN Shan;LIU Bing;SONG Lu-Yao;WANG Si-Yi;LI Jing-Jing(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China;School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China;Shanghai General Hospital,Shanghai 200080,China;School of Medicine,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学药学院,上海200240 [2]上海交通大学农业与生物学院,上海200240 [3]上海市第一人民医院,上海200080 [4]上海交通大学医学院,上海200240

出　　处：《中国生物化学与分子生物学报》2022年第10期1390-1402,共13页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by National Natural Science Foundation of China(No. 81773618);Shanghai Jiao Tong University Medical-Industrial Crossover Research Program (No. ZH2018QNA13)。

摘　　要：趋化因子及其受体信号通路是肿瘤细胞转移的主要调控因素之一,趋化因子受体CXCR4和XCR1都被证明参与了乳腺癌的进展。本文基于膜蛋白酵母双杂交发现了XCR1-CXCR4这一尚未报道过的相互作用对,进一步通过生物发光共振能量转移技术(bioluminescence resonance energy transfer,BRET)验证并发现XCR1可以竞争性地结合CXCR4受体(P<0.01),形成异源二聚体。在功能方面,首先通过XCR1和CXCR4瞬时转染HEK293细胞进行划痕实验,加入30 nmol/L SDF-1β后,共转组41.55%的伤口愈合率低于单转CXCR4组的58.75%,说明XCR1的共表达抑制了基质细胞衍生因子-1β(SDF-1β)/CXC趋化因子受体4型(CXCR4)信号通路介导的细胞运动性(P<0.05);其次,利用CXCR4-EGFP转基因HEK293细胞系,共表达XCR1后,流式细胞术检测细胞表面CXCR4受体荧光。结果显示,在30 nmol/L SDF-1β的诱导下,XCR1能够加速异源二聚体中CXCR4的内化(P<0.05),使得内化率从14.38%上升到64.10%;最后,分别检测了控制细胞增殖的Akt和控制细胞迁移的ERK信号通路的变化。结果发现,在SDF-1β刺激10 min后,单转CXCR4组的ERK磷酸化为3.59倍,而共转染XCR1/CXCR4组ERK的磷酸化水平仅为2.08倍,二聚化使得ERK磷酸化水平下降,且激活时间缩短;而Akt的磷酸化水平几乎不受影响。本研究揭示了CXCR4和XCR1二聚化现象,以及该二聚体对CXCR4介导的细胞运动性、受体内化和ERK磷酸化的影响。提示靶向XCR1的药物可以成为CXCR4交叉脱敏的候选药物,对于抑制乳腺癌转移提供了一个可供选择的思路。Chemokine signal pathways are important for the regulation of tumour metastasis.Chemokine receptors CXCR4(C-X-C chemokine receptor type 4) and XCR1(chemokine XC receptor 1) are involved in the metastasis of breast cancer,while the interaction between them remains unclear.Here we first identified the interaction between CXCR4 and XCR1 based on membrane protein yeast two-hybrid assays.Bioluminescence resonance energy transfer(BRET) showed that XCR1 could competitively bind to CXCR4 to form a heterodimer(P < 0.01).Results of wound healing assays via transient transfection of XCR1 and CXCR4 into HEK293 cells showed that 41.55% of the migration area rate in the co-transformation group was lower than 58.75% in the CXCR4-alone group after adding 30 nmol/L SDF-1β.The co-expression of XCR1 inhibited the cellular motility,possibly mediated by the SDF-1β(stromal cell-derived factor 1)/CXCR4 signal pathway(P < 0.05).Furthermore,CXCR4 on the cell surface after co-expression of XCR1 in CXCR4-EGFP transgenic HEK293 cells was detected by flow cytometry.And the result suggested that XCR1 could accelerate the internalization of CXCR4 into the heterodimer induced by 30 nmol/L SDF-1β(P<0.05),which increased the internalization rate from 14.38% to 64.10%.Finally,the phosphorylation of Akt and ERK,which were involved in cell proliferation and migration,respectively,were examined.After 10 minutes of SDF-1β stimulation,ERK phosphorylation in the CXCR4-alone group showed a 3.59-fold increase,whereas the increase of ERK phosphorylation in the co-transfected group was only 2.08-fold.Interestingly,heterodimer formation reduced the phosphorylation level of ERK and shortened the activation time,whereas the phosphorylation level of Akt remained unchanged.Collectively,our findings revealed the hetero-dimerization of CXCR4 and XCR1 and its effects on CXCR4-mediated cellular motility,receptor internalization,and ERK pathway phosphorylation.Therefore,XCR1-targeting drugs could be candidates for cross-desensitization of CXCR4 and might represen

关 键 词：趋化因子 异源二聚化 CXC趋化因子受体4型 趋化因子XC受体1 基质细胞衍生因子-1β 生物发光共振能量转移技术 

分 类 号：Q51[生物学—生物化学]