基于μ阿片受体羧基端^(375)STANT^(379)磷酸化位点的多肽干预对吗啡介导的受体下游信号通路的影响  

EFFECT OF PEPTIDE INTERVENTION BASED ON THE CARBOXYL-TERMINAL ^(375)STANT^(379) PHOSPHORYLATION SITE OF μ OPIOID RECEPTOR ON THE MORPHINE-MEDIATED RECEPTOR DOWNSTREAM SIGNALING PATHWAY

作　　者：李笑妍 周筱慧 董河[1] 董铭心 LI Xiaoyan;ZHOU Xiaohui;DONG He;DONG Mingxin(Department of Anesthesiology,The Affiliated Hospital of Qingdao University,Qingdao 266075,China)

机构地区：[1]青岛大学附属医院麻醉科,山东青岛266075 [2]青岛大学药学院

出　　处：《精准医学杂志》2023年第4期310-314,共5页Journal of Precision Medicine

基　　金：国家自然科学基金面上项目(81873729)。

摘　　要：目的探讨基于μ阿片受体(MOR)羧基端^(375)STANT^(379)磷酸化位点的多肽TAT-Q368-T379干预,对μ阿片受体下游信号通路的影响。方法将HA-MOR质粒与pGloSensorTM-22F质粒共转染的HEK-293细胞分为空白组(A组)、DAMGO组(B组)、吗啡组(C组)、0.5×10^(-5)mol/L多肽TAT-Q368-T379与吗啡联用组(D组)、10-5mol/L多肽TAT-Q368-T379与吗啡联用组(E组)、2×10^(-5)mol/L多肽TAT-Q368-T379与吗啡联用组(F组),通过GloSensor cAMP生物传感器测定各组细胞中的cAMP含量。将MOR-C端标记Nanoluc的质粒以及β-arrestin2-N端标记EYFP的质粒共转染完成的HEK-293细胞分为空白组(G组)、DAMGO组(H组)、吗啡组(I组)、10^(-5)mol/L多肽TAT-Q368-T379与吗啡联用组(J组)、3×10^(-5)mol/L Cmpd101与吗啡联用组(K组),通过蛋白质相互作用分析法测定各组细胞MOR激动后对β-arrestin2的募集效应。将HA-MOR质粒转染完成的HEK-293细胞分为空白组(L组)、吗啡组(M组)、10^(-5)mol/L多肽TAT-Q368-T379与吗啡联用组(N组)以及3×10^(-5)mol/L Cmpd101与吗啡联用组(O组),通过细胞免疫荧光染色法检测各组细胞的受体平均荧光强度。结果GloSensor cAMP生物传感器检测结果显示,C~F组细胞EC_(50)值差异有显著性(F=13.12,P<0.05),其中D、E、F组EC_(50)值较C组均显著减小(P<0.05)。蛋白质相互作用分析法分析显示,I~K组细胞E_(max)值差异有显著性(F=185.95,P<0.05),其中J、K组E_(max)值较I组均显著减小(P<0.01)。细胞免疫荧光染色法染色结果显示,L~O组细胞表面受体荧光强度比较差异有显著性(F=17.46,P<0.05),其中与M组比较,N、O组细胞表面受体荧光强度均显著增高(P<0.01)。结论基于MOR羧基端^(375)STANT^(379)磷酸化位点设计的多肽TAT-Q368-T379可有效增强吗啡介导的MOR下游G蛋白信号通路的激活,减少吗啡介导的β-arrestin2招募,从而减少由吗啡介导的MOR的内吞。Objective To study the effect of peptide TAT-Q368-T379 intervention based on the carboxyl-terminal ^(375)STANT^(379) phosphorylation site ofμopioid receptor(MOR)on the MOR downstream signaling pathway.Methods HEK-293 cells co-transfected with HA-MOR plasmids and pGloSensorTM-22F plasmids were divided into blank group(group A),DAMGO group(group B),morphine group(group C),0.5×10^(-5) mol/L peptide TAT-Q368-T379 combined with morphine group(group D),10^(-5) mol/L peptide TAT-Q368-T379 combined with morphine group(group E),and 2×10^(-5) mol/L peptide TAT-Q368-T379 combined with morphine group(group F).The GloSensor cAMP biosensor was used to measure cAMP content in each group.HEK-293 cells co-transfection with plasmids labeled with Nanoluc at the MOR-C terminal and plasmids labeled with EYFP at theβ-arrestin2-N terminal were divided into blank group(group G),DAMGO group(group H),morphine group(group I),10^(-5) mol/L peptide TAT-Q368-T379 combined with morphine group(group J),and 3×10^(-5) mol/L Cmpd101 combined with morphine group(group K).Protein interaction analysis assay was used to determine the recruitment of β-arrestin2 after MOR activation in each group.HEK-293 cells transfected with HA-MOR plasmids were divided into blank group(group L),morphine group(group M),10^(-5) mol/L peptide TA-Q368-T379 combined with morphine group(group N),and 3×10^(-5) mol/L Cmpd101 combined with morphine group(group O).Live cell immunofluorescence staining assay was used to determine the mean fluorescence intensity of receptors in each group.Results The results of GloSensor cAMP biosensor showed that the median effective concentration(EC_(50))values of groups C to F were significantly different(F=13.12,P<0.05),and the EC_(50) values in groups D,E,and F were significantly decreased compared with group C(P<0.05).The results of protein interaction analysis assay showed that the E_(max) values of groups I to K were significantly different(F=185.95,P<0.05),and the E_(max) values of groups J and K were significantly decreased comp

关 键 词：受体 阿片样 μ 磷酰化 吗啡 肽类 信号传导 GTP结合蛋白质类 β-抑制蛋白2 

分 类 号：R971.2[医药卫生—药品]