G 蛋白偶联受体激酶 6 的功能性核定位信号研究

In addition to the role in phosphorylation of G protein-coupled receptors (GPCRs) at the plasma membrane, which desensitizes the GPCR signaling, G protein-coupled receptor kinases (GRKs) have recently been found to act to regulate several important nuclear factors. However, the mechanism of the GRKs target to the nucleus is largely unknown. We have reported previously that GRK6 is capable of shuttling between plasma membrane and nucleus in a condition of depalmitoylation. With protein sequence swapping technique, we have recently found that two fragments (64 amino acids residues in total) from RGS domain of GRK6 could translocate the cytoplasmic GRK4 protein into nucleus, suggesting that there are nuclear localization regulating signals in these regions. Based on the novel discovery of potential nuclear localization regulating signals in GRK6, this proposal will focus on: (1) verification of the depalmitoylation-dependent nuclear translocation of GRK6 in living cells, (2) screening of the nuclear localization sequences (NLS) in the nuclear GRK4-GRK6 chimera, (3) identification and characterization of nuclear localization regulating signals in GRK6, and (4) determination of kinase activities and DNA-binding abilities of both GRK6 and its NLS mutants. This proposal will reveal the nuclear localization regulating signals in GRK6, which will provide insights into understanding the mechanism of nuclear shuttling of GRKs. It will also forge a path in search of potential therapeutic targets in GRK signaling.

近年发现，G 蛋白偶联受体激酶（GRK）除磷酸化胞膜 G 蛋白偶联受体（GPCR）、去敏GPCR的信号转导作用外，还具有直接调控核内生物分子的重要功能，但目前对GRK的核定位机制几乎一无所知。我们曾经报道GRK6在脱棕榈酰化条件下从胞膜转运至胞核，最近采用序列交换技术进一步发现GRK6分子RGS结构域的2个片段共64个氨基酸残基能够将胞浆蛋白GRK4全部转入细胞核，提示该区域隐藏功能性核定位调控信号。本项目拟在此基础上：（1）进一步确证GRK6分子的脱棕榈酰化依赖的胞核转运现象；（2）筛选该GRK4-GRK6嵌合分子中的核定位关键序列；（3）鉴定GRK6的功能性核定位调控信号；（4）检测核定位调控位点对 GRK6 激酶活性及DNA结合能力的影响。 本项目揭示GRK6的核定位调控机制，为后续开展GRK6的胞核信号转导研究、寻找新的GRK信号通路干预靶点提供理论依据。

G蛋白偶联受体激酶(G protein-coupled receptor kinases，GRKs)磷酸化被配体激活的G蛋白偶联受体（G protein-coupled receptors，GPCRs），负性调控GPCRs信号通路。新近的研究发现，某些GRKs分子如GRK6 等通过磷酸化或非磷酸化的方式直接调控细胞核内的底物分子。定位于细胞核是GRKs直接调控胞核内底物的前提，但目前对包括GRK6 在内的GRKs分子的胞核定位机制仍不清楚。本项目从筛选GRK6 的核定位序列( Nuclear localization sequences，NLSs)及其关键基团和介导胞核转运的核转运受体二个方面揭示GRK6核定位调控机制。通过构建系列缺失突变子初筛GRK6 的NLSs、以及对初筛得到的NLS区域的碱性氨基酸进行点突变发现，GRK6分子中 K389、K390、K391碱性氨基酸的单个位点突变显著抑制GRK6胞核定位能力，这些位点的突变均未破坏GRK6 对内源性毒蕈碱M3 受体介导的细胞内钙离子释放的抑制作用；采用RNA干扰技术分别沉默候选的十个核转运受体（Importin，IPO）筛选发现，沉默Importin 11显著减弱GRK6 的胞核定位能力；双分子荧光互补实验及免疫共沉淀实验检测到GRK6 与IPO11 存在相互作用的信号。研究结果表明，K389、K390和 K391是GRK6去棕榈去酰化下胞核定位所必需的关键基团，突变这些关键基团未破坏其激酶活性， GRK6的胞核转运受到Impotin 11）的直接调控 。该项目的完成，为进一步揭示GRK6胞核转运机制及其功能提供了有价值的信息。