蛋白酶激活接受器ㄧ與下游G蛋白α次單元及細胞外信號調節激酶在富含窖蛋白的分層中位置與分布之研究

Localization and Distribution of PAR1, Downstream G Protein α Subunits, and ERK in the Caveolin-enriched Fractions

許哲源(Che-Yuan Hsu)

指導教授 ： 傅化文

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2006年)

摘要

蛋白酶激活接受器一(protease-activated receptor 1)是一種G蛋白連結接受器(GPCR)，可以直接連結G蛋白α q/11次單元、G蛋白α i2次單元、G蛋白α 12/13次單元傳遞訊息。當其胺基端的特定序列被不可逆的切除活化後，蛋白酶激活接受器一經由克林斯林(clathrin)所進行的內吞作用(endocytosis)，進入細胞並運送到溶酶體(lysosome)進行降解。窖蛋白一(caveolin-1)是細胞膜上細微坑洞(caveolae)中富含的主要結構蛋白，並且在調控訊息傳遞和G蛋白連結接受器的內吞中扮演重要角色。然而目前對於蛋白酶激活接受器一以及其下游G蛋白alpha次單元和細胞外信號調節激酶(ERK)在蛋白酶激活接受器一活化前後是否位於細胞膜上細微坑洞並不清楚。在這項研究中，經由無界面活性劑(detergent-free)蔗糖梯度離心法製備富含窖蛋白一的分層來檢測這些蛋白質的分佈。在蛋白酶激活接受器一活化之前，此接受器幾乎都位於細胞膜上細微坑洞中。且在富含窖蛋白一的分層中發現了大量的G蛋白α q次單元和G蛋白α 13次單元。在高密度分層中發現大部分的G蛋白α i次單元、G蛋白α 12次單元和細胞外信號調節激酶。而蛋白酶激活接受器一活化之後，此接受器從富含窖蛋白一的分層離開並且降解。G蛋白α q次單元和G蛋白α 13次單元於富含窖蛋白一的分層中並不移動。G蛋白α i次單元移入但之後移出了富含窖蛋白一的分層，而G蛋白α 12次單元逐漸移出了富含窖蛋白一的分層。此外，也發現在第四到第六以及第七到第十二分層中有磷酸化的細胞外信號調節激酶分布，在此同時，細胞外信號調節激酶在富含窖蛋白一的分層中逐漸累積。這些結果指出脂筏(lipid rafts)和細胞膜上細微坑洞也許可作為細胞膜的微區域(microdomain)，使得蛋白酶激活接受器一在此微區域中調控它下游的訊息傳遞。

關鍵字

蛋白酶激活接受器 ； 細胞膜上細微坑洞 ； 窖蛋白 ； 脂筏 ； G蛋白連結接受器 ； 凝血蛋白酶

並列摘要

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) activated by thrombin, directly couples with Gαq/11, Gαi2, Gα12/13 to transduce signals. After activation by irreversible cleavage at the specific site of its N-terminus, PAR1 was internalized via clathrin-dependent endocytosis and sorted to lysosomes for degradation. Caveolin-1, a major structure protein enriched in caveolae, plays important roles in regulation of signal transduction and internalization of GPCRs. However, it is not clear whether PAR1, its downstream G protein alpha subunits, and extracellular signal-regulated kinase (ERK) reside in caveolae before and after the receptor activation. In this study, the caveolin-enriched fractions prepared by the detergent-free sucrose gradient centrifugation were used to examine the distribution of these proteins. Before PAR1 activation, the receptor was highly confined in the caveolin-enriched fractions. Gαq and Gα13 were predominantly found in the caveolin-enriched fractions. A large portion of Gαi, Gα12, and ERK was observed in the high-density fractions. After PAR1 activation, the receptor was moved out of the caveolin-enriched fractions to be degraded. Gαq and Gα13 were still immobilized in the caveolin-enriched fractions. Gαi was moved in and then moved out of the caveolin-enriched fractions, whereas Gα12 was gradually moved out of the caveolin-enriched fractions. Furthermore, phosphorylated ERK was found in the fraction 4 to 6 and 7 to 12, while ERK was gradually accumulated in the caveolin-enriched fractions. These results indicate that lipid raft/caveolae may act as major plasma membrane microdomains where PAR1 is localized to modulate its downstream signal transduction.

並列關鍵字

PAR ； caveolae ； caveolin ； lipid raft ； GPCR ； Thrombin

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