蛋白激酶D1通过磷酸化多巴胺D2受体促进可卡因成瘾发展的作用与机制研究

Cocaine addiction is a serious behavioral disorder, but drugs for addiction always have some side effects. It is generally accepted that D2R is a key molecule involved in the development of cocaine addiction. It might be a new strategy for the development of drugs based on the regulatory proteins of D2R, which can interfere with the function of D2R. Our lab pre-experiments suggested that knockdown of PKD1 in rat dorsal striatum significantly reduced locomotors activity induced by cocaine, and PKD1 can reduce the D2R membrane localization by phosphorylating D2R. Based on our results, we hypothesize that activation of PKD1 can reduce D2R membrane localization, block D2R signaling pathways and thus promote the progression of cocaine addiction. This project will inject peptides into rat dorsal stritum or nucleus accumbens to block the phosphorylation of D2R mediated by PKD1 to study the locomotors activity and conditioned place preference when rats injected cocaine. Meanwhile we interface the phosphorylation of D2R by PKD1 in HEK 293T cell line and cultured primary striatal neurons to study the underlying mechanisms. This may help to elucidate the mechanism of cocaine addiction, and provide a promising candidate for pharmacotherapeutic intervention of DA signaling for the treatment of drug addiction.

可卡因成瘾是一种严重的行为疾病，目前关于可卡因成瘾的药物效果欠佳或有严重副作用。D2R是公认的负向调控可卡因成瘾的关键分子，以D2R为研究对象，找出D2R新的调节蛋白，为设计新药物提供思路显得尤为重要。本课题组前期研究发现敲低大鼠背侧纹状体PKD1明显降低可卡因所引起的运动活性， PKD1可以磷酸化D2R，降低D2R细胞膜定位，抑制D2R活性。据此我们推测PKD1的激活可以降低D2R细胞膜定位，抑制D2R活性，阻断其介导的信号通路，促进可卡因成瘾的发展。本课题拟用大鼠脑内特定区域给药进行可卡因所诱导的运动活性及条件性位置偏好实验，观察纹状体或伏隔核内干扰PKD1磷酸化D2R后，大鼠对可卡因的反应。同时在HEK293T细胞系及大鼠原代纹状体神经元干扰PKD1对D2R的磷酸化，观察D2R的细胞分布及信号通路的变化。通过上述实验以期阐明可卡因成瘾的发生机制，为药物的研制提供新的思路。

D2R是公认参与可卡因成瘾的关键分子。以D2R为靶点的药物前景良好，但目前关于可卡因成瘾的药物效果欠佳或有严重的副作用。以D2R为研究对象，找出D2R新的调节蛋白，为设计药物提供新的思路显得尤为重要。本项目课题组发现大鼠背侧纹状体中PKD1蛋白的敲低能够减弱可卡因所诱导的运动活性增加的现象。体外激酶实验发现PKD1可以磷酸化D2R 365位的丝氨酸，并通过这种磷酸化降低D2R的细胞膜定位，进而增强D2R所介导的下游细胞外信号调节激酶（extracellular signal-regulated kinase, ERK）的活性。更重要的是我们设计了一种干扰肽Tat-S365，用于干扰PKD1对D2R的磷酸化。将干扰肽Tat-S365注射到大鼠背侧纹状体中能够明显抑制可卡因所诱导的大鼠运动活性升高的现象。因此以D2R 365位丝氨酸为药物靶点开发药物可以用于治疗可卡因成瘾或其他因多巴胺功能异常所引起的疾病。