猪生长激素与其受体复合体（GH-GHR）核转运分子机制的研究

Traditional theory thinks that GH binding to its receptor expressed on the target cell surface shows its physiological roles by activation of intracellular signaling pathway. However, recent studies have indicated that GH-GHR complex localized in the nuclear also possess important physiological functions. But until now, the nuclear translocation mechanism(s) of GH-GHR complex remains unclear. Our research group previously found that IMPα/β is required for the nuclear translocation of GH-GHR complex, and that the nuclear translocation of GH-GHR complex is dramatically inhibited after blocking early endosome or Bip that specifically localized in endoplasmic reticulum (ER). On the basis of the above-mentioned results, we speculate that there are two mechanisms by which GH-GHR complex translocates into the nuclear: 1) the nuclear translocation of GH-GHR complex is mediated by endosome under the participation of importin and nucleoporin; 2) GH-GHR complex transports into cytoplasm via ERAD pathway which is mediated by Bip and Sec61, and then, GH-GHR translocates into the nuclear under the participation of importin and nucleoporin. The techniques of Live cell imaging system, Immunoelectron microscopy and Confocal laser scanning microscope in combination of methods of specific inhibitor, SiRNA, antibody neutralization, separation of cell organelles and cytoplasm replacement will be used to verify the above-mentioned hypothesis in the model of porcine hepatocytes and CHO transfected with porcine GHR. The research results will establish molecular basis for in-depth study of physiological roles of the nuclear-localized GH-GHR complex.

传统认为GH与细胞膜上GHR结合后激活胞内信号通路而发挥生理作用，然而新近发现GH-GHR复合体转运至细胞核同样具有重要生理功能，但其核转运的分子机制尚未阐明。课题组前期发现IMPα/β参与了GH-GHR复合体核转运，还发现阻断内体或内质网中Bip可显著抑制GH-GHR复合体的核转运。由此我们推测GH-GHR复合体存在两种核转运机制：1）GH-GHR复合体以内体作为载体并在核转运蛋白及核孔蛋白参与下转入细胞核；2）GH-GHR复合体经内质网中Bip与Sec61介导的ERAD途径进入细胞质，然后在核转运蛋白与核孔蛋白参与下转入细胞核。为此，本项目拟以猪肝细胞并辅以猪GHR转染细胞系为模型，综合运用活细胞工作站、电镜、共聚焦显微镜与免疫共沉淀等技术并结合特异性抑制剂、SiRNA、抗体中和、细胞器分离及胞质液替换等方法论证上述推测。研究结果将为深入探究GH-GHR复合体核定位的生理作用奠定基础。

传统理论认为 GH 与细胞膜表面 GHR 结合后激活胞内信号通路而发挥生理作用。然而新近研究发现，GH-GHR 复合体转运至细胞核同样具有重要的生物学功能。但其核转运的分子机制尚未阐明，是一个亟待解决的重要科学问题。因此，本研究旨在明确GH/GHR核定位的分子机制。为此，在国家自然基金的资助下，我们开展了如下几个方面的研究：①综合运用活细胞工作站、 激光共聚焦显微镜等技术研究 Clathrin 或 Caveolin 介导的 GH-GHR 复合体的内化与其核定位的关系；②综合运用免疫共沉淀与激光共聚焦显微镜技术分析GH-GHR 或 G120R-GHR 复合体与 IMPα/β 的对接情况， 验证 GH-GHR 复合体与 IMPα/β 的结合方式；③运用电镜与免疫共沉淀技术分析早期内体中的 GH-GHR 复合体与IMPα/β 的结合情况，综合运用电镜、 激光共聚焦显微镜与免疫共沉淀技术验证早期内体中 GH-GHR 复合体在 IMPα/β 介导下与核孔蛋白 Nup358 的结合情况，最终阐明了内体介导的 GH-GHR 复合体核转运的分子机制；④ 综合采用共定位分析、免疫电镜及免疫共沉淀等技术方法，研究内质网中 Bip 与 Sec61 介导的 GH-GHR 复合体核转运的分子机制。我们的研究揭示了猪GH-GHR复合体核转运的基本分子机制，发现了GH-GHR核定位的分子路径，具体发现如下，分子路径①：猪GH与GHR在细胞表面结合后，GH-GHR在clathrin或者cavelion的介导下复合体内吞进入细胞质，然后内化进入Rab5阳性内体，随即转运至内质网并与内质网上易位子Sec61结合，后经ERAD途径再进入细胞质并与核转运蛋白IMPα/β结合，并在其介导下与核孔复合物NUP358结合互作，并在其介导下进入细胞核；分子路径②：还发现另一条猪GH-GHR核转运的路径，即“内体”路径。猪GH-GHR复合体通过Clathrin/Cavelolin介导的内吞作用进入细胞质，随即转入NAE（Nuclear envelope-associated endosomes）中，并在其介导下与核膜对接，而后进入细胞核。揭示GH/GHR核转运的路径为深入研究GH/GHR的互作模式及生理功能奠定基础；对畜牧业生产也具有间接的指导作用；此外，对相关生长因子及细胞因子的研究也具有重要的提示与借鉴意义。