本文關(guān)鍵詞：煙酸受體GPR109A信號轉(zhuǎn)導(dǎo)和內(nèi)吞分子機制研究　出處：《浙江大學(xué)》2011年博士論文　論文類型：學(xué)位論文

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【摘要】：煙酸作為一種降脂藥物已經(jīng)在臨床上廣泛使用了50多年,煙酸不僅能夠有效降低LDL-C的同時,還能提高HDL-C的水平,而煙酸受體GPR109A的發(fā)現(xiàn)為治療高血脂和心血管系統(tǒng)疾病提供了一個很好的分子靶標(biāo),GPR109A受體信號轉(zhuǎn)導(dǎo)機制的研究和小分子激動劑藥物的研發(fā)因而受到廣泛的重視。然而GPR109A受體內(nèi)吞和其介導(dǎo)的信號轉(zhuǎn)導(dǎo)的詳細(xì)機制還不清楚。 研究表明GPR109A受體的內(nèi)吞主要由GRK2和arrestin3調(diào)節(jié),而G_(βγ)起到了招募GRK2到細(xì)胞膜上的作用。蔗糖預(yù)處理或siRNA干擾網(wǎng)格蛋白的表達(dá),GPR109A受體的內(nèi)吞均受到顯著抑制,說明其內(nèi)吞是網(wǎng)格蛋白小泡依賴性的。進(jìn)一步研究表明,當(dāng)配體去除后,GPR109A能迅速回到細(xì)胞膜上,而且內(nèi)含體的酸化對這一過程并不是必需的。百日咳毒素預(yù)處理不僅能抑制煙酸對forskolin引起的胞內(nèi)cAMP含量升高的抑制,還能抑制煙酸介導(dǎo)的胞內(nèi)鈣流以及GPR109A受體的內(nèi)吞。 通過缺失和定點突變,我們發(fā)現(xiàn)GPR109A羧基端在調(diào)控受體從內(nèi)質(zhì)網(wǎng)轉(zhuǎn)運到細(xì)胞膜上,以及受體內(nèi)吞、失敏及其自身活化方面扮演非常重要的角色。△295-314突變體完全不能定位到細(xì)胞膜上而位于內(nèi)質(zhì)網(wǎng)中,而且不會介導(dǎo)激動劑引發(fā)的信號轉(zhuǎn)導(dǎo)。△315-328突變體介導(dǎo)的信號轉(zhuǎn)導(dǎo)與野生型(WT)的受體相似,但其內(nèi)吞和失敏卻受到顯著的影響。通過逐步定點突變,我們發(fā)現(xiàn)絲氨酸和蘇氨酸簇(326STS328)在受體的內(nèi)吞、失敏以及arrestin3轉(zhuǎn)運到細(xì)胞膜上起到關(guān)鍵的調(diào)控作用。△329-343突變體與野生型GPR109A相似,配體刺激后會快速發(fā)生內(nèi)吞,然而出乎意料的是,該突變交體還能通過配體非依賴性的途徑發(fā)生內(nèi)吞。 幾乎所有的GPCR都會通過MAPK傳遞信號,MAPK是重要的細(xì)胞調(diào)控因子,調(diào)控許多重要的生理過程如細(xì)胞的生殖、生長、分化、凋亡等。利用細(xì)胞外源性表達(dá)GPR109A的CHO-K1以及內(nèi)源性表達(dá)GPR109A的A431細(xì)胞,我們發(fā)現(xiàn)GPR109A能介導(dǎo)ERK1/2快速的,PTX敏感的磷酸化。通過時間梯度研究,我們發(fā)現(xiàn)PKC激酶主要調(diào)控GPR109A介導(dǎo)的早期ERK1/2的活化,而EGFR轉(zhuǎn)激活則參與整個GPR109A介導(dǎo)的ERK1/2磷酸化。通過過量表達(dá)G_(βγ)拮抗劑PARK1-CT and a-transducin,我們發(fā)現(xiàn)G_(βγ)在調(diào)控GPR109A介導(dǎo)的ERK1/2活化過程中起到非常重要的調(diào)控作用。另外,利用arrestin-2/3SiRNA特異性沉默穩(wěn)定表達(dá)GPR109A的HEK293細(xì)胞內(nèi)arrestin-2/3蛋白,我們發(fā)現(xiàn)arrestin-2/3并不參與調(diào)控GPR109A介導(dǎo)的ERK1/2的活化。 通過上述研究,我們闡明了人煙酸受體GPR109A受體內(nèi)吞的詳細(xì)機制,其羧基端對受體定位、內(nèi)吞、失敏、irrestin3結(jié)合以及自身活化的調(diào)控,以及其調(diào)控ERK1/2磷酸化的途徑。在此基礎(chǔ)之上,我們希望通過后續(xù)實驗進(jìn)一步闡明GPR109A介導(dǎo)的降低血脂和引起皮膚潮紅的詳細(xì)機理,為開發(fā)更加高效而同時不會引起皮膚潮紅的降血脂藥物提供理論基礎(chǔ)。
[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 緯) 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 緯) 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R96;R341

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 楊靜文;家蠶corazonin受體信號轉(zhuǎn)導(dǎo)機制及生理功能的研究[D];浙江大學(xué);2013年

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本文編號：1368267