目的 A20结合的核因子抑制蛋白(ABIN1蛋白)是通过细菌双杂交技术筛选得到的与MOR羧基端(C-端)有相互作用的蛋白。本研究在确定ABIN1蛋白与μ阿片受体(MOR)相互作用的基础上,明确ABIN1对MOR受体激动活性及MOR激活后信号通路的影响。方法 (1)用GST-pull-down实验、免疫荧光细胞化学共定位及免疫共沉淀(Co-IP)实验验证ABIN1与MOR-C有无相互作用。(2)建立稳定转染ABIN1与MOR的CHO细胞株,用[3H]diprenorphine进行受体配体结合实验,观察ABIN1对MOR与配体结合力的影响。(3)用[35S]GTP-γS实验,观察ABIN1对MOR激动活性的影响。(4)通过SDS-PAGE电泳观察ABIN1对MOR激活后受体磷酸化及ERK/p-ERK水平的影响,通过cAMP含量测定确定ABIN1对DAMGO急性作用于MOR抑制cAMP作用的影响。结果 GST-pulldown实验在细胞外证明ABIN1与MOR-C有相互作用,免疫荧光细胞化学共定位实验表明ABIN1与MOR在胞膜存在共定位,Co-IP实验证明在细胞水平ABIN1与MOR有相互作用。[3H]diprenorphine受体配体结合实验表明ABIN1对MOR受体配体亲和力没有明显影响,[35S]GTP-γS实验证明ABIN1能够抑制激动剂对MOR的激动活性,EC50值显著增加(P<0.05)。量效曲线右移。ABIN1能够明显抑制DAMGO作用于MOR后对cAMP含量的降低作用(P<0.05),ABIN1明显抑制MOR的磷酸化(P<0.05),ABIN1对MOR激活后的p-ERK水平有明显的抑制作用(P<0.01),对ERK水平没有明显影响。结论 ABIN1与MOR相互作用后抑制MOR受体激动活性、MOR的磷酸化及受体后信号通路。 Objective A20-bound nuclear factor inhibitor (ABIN1) is a protein that interacts with the carboxy-terminal (C-terminal) of MOR through bacterial two-hybrid screening. In this study, ABIN1 protein and mu opioid receptor (MOR) interaction on the basis of a clear ABIN1 on MOR receptor agonistic activity and MOR signaling pathway after activation. Methods (1) GST-pull-down assay, immunofluorescence cytochemistry co-localization and co-immunoprecipitation (Co-IP) experiments to verify whether ABIN1 and MOR-C interaction. (2) CHO cell lines stably transfected with ABIN1 and MOR were established, and receptor binding experiments with [3H] diprenorphine were performed to observe the effect of ABIN1 on the binding ability of MOR to ligands. (3) [35S] GTP-γS experiment was used to observe the effect of ABIN1 on MOR agonistic activity. (4) The effect of ABIN1 on MOR and phosphorylation of ERK / p-ERK after MOR activation was observed by SDS-PAGE electrophoresis. The effect of ABIN1 on the inhibition of cAMP induced by MOR was determined by cAMP assay. Results The GST-pulldown assay demonstrated that ABIN1 and MOR-C interacted extracellularly. Immunofluorescence cytochemistry co-localization experiments showed that ABIN1 and MOR co-localized in the cell membrane. Co-IP experiments demonstrated that ABIN1 interacts with MOR at the cellular level . The [3H] diprenorphine receptor ligand binding assay showed that ABIN1 had no significant effect on the MOR receptor ligand affinity. [35S] GTP-γS assay demonstrated that ABIN1 inhibited agonist activity to MOR with a significant increase in EC50 (P <0.05 ). The efficiency curve shifts to the right. ABIN1 could significantly inhibit the decrease of cAMP content after DAMGO treatment (P <0.05), ABIN1 significantly inhibited the phosphorylation of MOR (P <0.05), and ABIN1 significantly inhibited the p-ERK level after MOR activation (P < P <0.01), no significant effect on ERK levels. Conclusion ABIN1 interacts with MOR and inhibits MOR receptor activity, phosphorylation of MOR and post-receptor signaling pathway.