逆膜运输对钙代谢相关受体的调节作用及相关分子机制研究

Metabolic bone diseases and other related diseases caused by calcium metabolism disorders have led to serious threat to human health. Three receptors including calcium sensing receptor(CaSR), parathyroid hormone receptor(PTHR) and calcitonin receptor(CTR) are G protein-coupled receptors that are crucial for calcium metabolism. However, mechanism underlying how their expression and activity at subcellular level are regulated remains unknown. Our recent study has shown that internalized CaSR、PTHR and CTR traffick upon substrate stimulation are regulated by the retrograde trafficking from endosome to TGN which may further facilitate their recycling back to cell surface. We also found that inactivation of Rab29, a novel Golgi associated small GTPase, led to dispersed distribution of the internalized receptors rather than with normal sorting to TGN. Together with these work, we hypothesized that internalized CaSR, PTHR and CTR are highly regulated by the conserved retrograde endosome-TGN trafficking pathway, in which Rab29 also plays an important role. Using the available molecular and cellular techniques, we will determine how the internalized CaSR, PTHR and CTR are regulated by Vps35, the key component protein of retromer complex. We will then study the role of Rab29 in regulating the subcellular localization and function of three receptors as well as the signaling process among retromer-Rab29 interactions. By studing the molecular mechanism of calcium metabolism, we will establish the theoretical foundation for the treatment of bone and other systems’ diseases caused by abnormal calcium metabolism and identify new drug targets that may lead to a new breakthrough for clinical treatment of these diseases.

钙代谢异常所致的骨代谢病及其它相关疾病严重危害人们的健康。钙敏感受体（CaSR）、甲状旁腺激素受体（PTHR）和降钙素受体（CTR）是一类与调节钙代谢密切相关的G蛋白偶联受体。然而这三种受体的活性在细胞水平上的分子调节机制尚未阐明。我们前期工作发现CaSR、PTHR和CTR内吞后能在传统逆膜运输机制的调节下经内体-反面高尔基体（TGN）途径被再循环重新发挥作用，而新发现的TGN蛋白Rab29功能的缺失会导致受体的逆向运输受阻。因此我们提出假设：CaSR、PTHR和CTR在内吞后能够通过逆膜运输机制从内体回到TGN上，并且这一过程受逆膜复合体-Rab29信号传导系统的调节。本课题拟运用分子细胞学的方法，深入阐明经典逆膜复合体介导的逆膜运输在CaSR、PTHR和CTR的代谢途径中所发挥的作用及Rab29对这一过程的调节机制，为治疗钙代谢异常引起的相关疾病提供基础理论依据和筛选小分子新药的靶点。

钙代谢相关受体（CaSR,PTHR,CTR）是一种与调节血清钙水平密切相关的G蛋白偶联受体，既往研究认为G蛋白偶联受体在激活后内吞进入细胞并最终被溶酶体降解，然而我们通过预实验发现钙代谢相关受体可以经内体运输至反式高尔基体（TGN）而实现受体循环利用，为了研究这一膜运输机制，我们通过构建相关标签质粒及突变基因质粒作为实验载体，运用细胞转染、免疫荧光染色、酶联免疫吸附测定、蛋白质印迹检测等技术方法，对通过小分子蛋白Rab29（Rab7L1）参与调控进而由逆膜运输体（Retromer）介导的钙代谢相关受体的逆膜运输机制进行了研究。首先我们通过使用特异性的ShRNA抑制Retromer的中心结合蛋白Vps35的活性后证实CaSR的核周定位及半衰期受Retromer调控，同时我们利用M6PR这一经典的逆膜运输模型研究发现，由Retromer介导的逆膜运输受Rab7L1的调节。进一步的挽救实验表明Rab7L1位于Retromer的下游发挥了对逆膜运输的调节作用，并且通过分析Rab7L1的亚细胞定位我们发现，其与TGN（而非内体、溶酶体及顺式高尔基体）具有显著的共定位特征。基于上述研究成果及预实验结果，我们探究了Rab7L1对CaSR，PTHR，CTR逆膜运输的调节作用，结果显示敲减Rab7L1导致CaSR及PTHR的逆膜运输受阻，并致使CaSR及PTHR的半衰期缩短以及胞内膜信号传导功能受阻，而CTR不受上述细胞内运输机制的影响。综上所述，Rab7L1作为Retromer的下游信号蛋白，参与调节了由Retromer所介导的CaSR及PTHR的逆膜运输，通过这一膜运输机制可将CaSR及PTHR在激活内吞入胞后从内体直接运输至反式高尔基体实现受体的循环利用，这与维持CaSR及PTHR的受体功能密切相关。本研究为治疗钙代谢异常引起的骨科及其他系统疾病奠定了基础理论依据，也为筛选小分子新药提供了候选靶点。