RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.
RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.
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DOI：
10.1111/jnc.12100
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发表时间：
2013-02
影响因子：
4.7
通讯作者：
Ko JL
中科院分区：
文献类型：
--
作者：
Nahar-Gohad P;Sultan H;Esteban Y;Stabile A;Ko JL
PCBP1是mu-阿片受体（MOR）基因的表达调节因子。我们假设存在一种PCBP1共调节因子，通过与PCBP1的蛋白-蛋白相互作用来调节人类MOR基因的表达。以PCBP1为诱饵，采用双杂交系统筛选人脑cDNA文库。RACK1蛋白含有7个WD结构域。PCBP1-RACK1相互作用通过双杂交系统在体内验证，以及与抗PCBP1抗体和人神经元NMB细胞裂解液共免疫沉淀，内源性表达PCBP1和RACK1。进一步的共免疫沉淀表明RACK1-PCBP1相互作用仅发生在细胞质中。单个和串行WD结构域缺失分析表明，RACK1的WD7是与PCBP1相互作用的关键结构域。RACK1过表达导致含有人MOR启动子和荧光素酶报告基因的p357质粒MOR启动子活性呈剂量依赖性降低。RT-PCR结果显示，RACK1 siRNA降低了NMB内源性RACK1 mRNA水平，升高了MOR mRNA水平。同样，RACK1的减少导致MOR蛋白的增加，通过3h -二丙诺啡结合实验证实。总的来说，本研究报告了RACK1的新作用，与PCBP1物理相互作用，参与调节神经元NMB细胞中人类MOR基因的表达。
PCBP1 is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein-protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. RACK1 protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 overexpression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by 3H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.
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DOI：
10.1073/pnas.0701065104
发表时间：
2007-04-03
影响因子：
11.1
作者：
Meng, Qingchang;Rayala, Suresh K.;Kumar, Rakesh
通讯作者：
Kumar, Rakesh
DOI：
10.1016/s0169-328x(03)00086-x
发表时间：
2003-04-10
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
Ko, JL;Liu, HC;Loh, HH
通讯作者：
Loh, HH
影响因子：
5.3
作者：
Chang, BY;Conroy, KB;Cartwright, CA
通讯作者：
Cartwright, CA
影响因子：
3.6
作者：
Choi, HS;Hwang, CK;Loh, HH
通讯作者：
Loh, HH
影响因子：
11.6
作者：
Nakashima, Ayako;Chen, Letian;Shimamoto, Ko
通讯作者：
Shimamoto, Ko