烟酸受体GPR109A信号转导和内吞分子机制研究

发布时间：2018-01-02 07:29

本文关键词：烟酸受体GPR109A信号转导和内吞分子机制研究　出处：《浙江大学》2011年博士论文　论文类型：学位论文

【摘要】：烟酸作为一种降脂药物已经在临床上广泛使用了50多年,烟酸不仅能够有效降低LDL-C的同时,还能提高HDL-C的水平,而烟酸受体GPR109A的发现为治疗高血脂和心血管系统疾病提供了一个很好的分子靶标,GPR109A受体信号转导机制的研究和小分子激动剂药物的研发因而受到广泛的重视。然而GPR109A受体内吞和其介导的信号转导的详细机制还不清楚。研究表明GPR109A受体的内吞主要由GRK2和arrestin3调节,而G_(βγ)起到了招募GRK2到细胞膜上的作用。蔗糖预处理或siRNA干扰网格蛋白的表达,GPR109A受体的内吞均受到显著抑制,说明其内吞是网格蛋白小泡依赖性的。进一步研究表明,当配体去除后,GPR109A能迅速回到细胞膜上,而且内含体的酸化对这一过程并不是必需的。百日咳毒素预处理不仅能抑制烟酸对forskolin引起的胞内cAMP含量升高的抑制,还能抑制烟酸介导的胞内钙流以及GPR109A受体的内吞。通过缺失和定点突变,我们发现GPR109A羧基端在调控受体从内质网转运到细胞膜上,以及受体内吞、失敏及其自身活化方面扮演非常重要的角色。△295-314突变体完全不能定位到细胞膜上而位于内质网中,而且不会介导激动剂引发的信号转导。△315-328突变体介导的信号转导与野生型(WT)的受体相似,但其内吞和失敏却受到显著的影响。通过逐步定点突变,我们发现丝氨酸和苏氨酸簇(326STS328)在受体的内吞、失敏以及arrestin3转运到细胞膜上起到关键的调控作用。△329-343突变体与野生型GPR109A相似,配体刺激后会快速发生内吞,然而出乎意料的是,该突变交体还能通过配体非依赖性的途径发生内吞。几乎所有的GPCR都会通过MAPK传递信号,MAPK是重要的细胞调控因子,调控许多重要的生理过程如细胞的生殖、生长、分化、凋亡等。利用细胞外源性表达GPR109A的CHO-K1以及内源性表达GPR109A的A431细胞,我们发现GPR109A能介导ERK1/2快速的,PTX敏感的磷酸化。通过时间梯度研究,我们发现PKC激酶主要调控GPR109A介导的早期ERK1/2的活化,而EGFR转激活则参与整个GPR109A介导的ERK1/2磷酸化。通过过量表达G_(βγ)拮抗剂PARK1-CT and a-transducin,我们发现G_(βγ)在调控GPR109A介导的ERK1/2活化过程中起到非常重要的调控作用。另外,利用arrestin-2/3SiRNA特异性沉默稳定表达GPR109A的HEK293细胞内arrestin-2/3蛋白,我们发现arrestin-2/3并不参与调控GPR109A介导的ERK1/2的活化。通过上述研究,我们阐明了人烟酸受体GPR109A受体内吞的详细机制,其羧基端对受体定位、内吞、失敏、irrestin3结合以及自身活化的调控,以及其调控ERK1/2磷酸化的途径。在此基础之上,我们希望通过后续实验进一步阐明GPR109A介导的降低血脂和引起皮肤潮红的详细机理,为开发更加高效而同时不会引起皮肤潮红的降血脂药物提供理论基础。
[Abstract]:Niacin, as a lipid lowering drug, has been widely used in clinic for more than 50 years. Niacin can not only effectively reduce LDL-C, but also improve the level of HDL-C. The discovery of nicotinic acid receptor GPR109A provides a good molecular target for the treatment of hyperlipidemia and cardiovascular diseases. The study of signal transduction mechanism of GPR109A receptor and the research and development of small molecular agonist drugs have attracted extensive attention. However, the detailed mechanism of GPR109A by swallowing in vivo and its mediated signal transduction is still unclear. Chu. Studies have shown that endocytosis of GPR109A receptors is mainly regulated by GRK2 and arrestin3. GSP (尾纬) played a role in recruiting GRK2 to the cell membrane. Sucrose pretreatment or siRNA interference with the expression of griddle protein was significantly inhibited in the endocytosis of GPR109A receptor. Further studies showed that GPR109A could rapidly return to the cell membrane when the ligand was removed. The pretreatment of pertussis toxin can not only inhibit the increase of intracellular cAMP induced by nicotinic acid. It also inhibited intracellular calcium flow mediated by nicotinic acid and endocytosis of GPR109A receptor. By deletion and site-directed mutation, we found that the carboxyl terminal of GPR109A regulates the transport of receptors from the endoplasmic reticulum to the cell membrane, as well as the uptake in vivo. Desensitization and its own activation play a very important role. 295-314 mutants can not be located on the membrane but in the endoplasmic reticulum. The signal transduction mediated by 315-328 mutants is similar to that of wild-type WTs. However, the endocytosis and desensitization of serine and threonine cluster 326STS328) were significantly affected by site-directed mutagenesis, and we found that serine and threonine cluster 326STS328) endocytosis. Desensitization and arrestin3 transport to the cell membrane play a key role in regulation. 329-343 mutant is similar to wild-type GPR109A and endocytosis occurs rapidly after ligand stimulation. Unexpectedly, however, the mutant could also develop endocytosis through ligand-independent pathways. Almost all GPCR signaling through MAPK is an important cellular regulatory factor, regulating many important physiological processes such as cell reproduction, growth, differentiation. Apoptosis. CHO-K1 expressing GPR109A and A431 cells expressing GPR109A were used. We found that GPR109A can mediate the rapid phosphorylation of ERK1/2 by time gradient. We found that PKC kinase mainly regulates the activation of early ERK1/2 mediated by GPR109A. EGFR transactivation is involved in the whole GPR109A mediated ERK1/2 phosphorylation. Antagonist PARK1-CT and a-transducin. We found that GSP (尾纬) plays a very important role in the regulation of ERK1/2 activation mediated by GPR109A. Arrestin-2/3SiRNA specific silencing was used to stably express arrestin-2/3 protein in HEK293 cells of GPR109A. We found that arrestin-2/3 is not involved in regulating the activation of ERK1/2 mediated by GPR109A. Through the above studies, we have elucidated the detailed mechanism of endocytosis of human nicotinic acid receptor GPR109A in vivo, and its carboxyl terminal targeting, endocytosis and desensitization of human nicotinic acid receptor GPR109A. The regulation of irrestin3 binding and self-activation, and its regulation of ERK1/2 phosphorylation. We hope to further elucidate the mechanism of GPR109A mediated reduction of blood lipids and skin flashes through follow-up experiments. To provide a theoretical basis for the development of more efficient and not cause skin flashes of antilipidemic drugs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R96;R341

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