阿拉伯芥 G 蛋白質訊息傳遞系統和 G 蛋白質訊息調節蛋白質 (AtRGS1) 分子機制探討

Abstract

異三元體 G 蛋白質系統是一個被研究相當透徹的主題，它在哺乳類動物和酵母菌的訊息傳遞及生理中扮演了許多重要角色。藉由配體的活化，G 蛋白質耦合性受器 (GPCR) 可促使 Gα 次元產生構形變化，並且引發在 Gα 之中 GTP 置換 GDP。活化狀態的 Gα (Gα-GTP) 會和 Gβγ 次元以及 GPCR 分離，且隨後在細胞內啟發一系列的訊息梯瀑反應。此訊息則會被 Gα 固有的 GTPase 酵素活性給終結，它可水解 GTP 回 GDP 並且將 Gα 恢復回基態。而 Gα 的 GTPase 活性可被 G 蛋白質訊息調控蛋白質 (RGS) 進一步地加速，它會和 Gα 交互作用並且關閉 Gα 的傳訊狀態。因為 GTP 被水解回 GDP，所以 Gα 會再和 Gβγ 重新組合在一起，以等待下一次刺激。 如同哺乳類動物，模式植物阿拉伯芥也擁有異三元體 G 蛋白質系統，且阿拉伯芥異三元體 G 蛋白質的各個成員參與了許多植物的生理功能，如荷爾蒙感受、細胞增殖及逆境抵抗。然而，和哺乳類動物 G 蛋白質廣泛的研究相比較，除了植物異三元體 G蛋白質的生理功能之外，該領域的科學家很少致力於其他方面的研究。因此在本研究之中，我們使用BODIPYTR-GTP 及 Lucifer yellow VS這兩種螢光探針，研究阿拉伯芥 Gα 次元 (AtGPA1) 和 RGS (AtRGS1) 的功能以及它們兩者之間的交互作用。此外，我們也篩選 AtGPA1 以及 AtRGS1 的 RGS 功能區塊的結晶條件。藉由使用 BODIPYTR-GTP，我們可即時監視到 AtRGS1 的 RGS 功能區塊專一地加速 AtGPA1 的 GTPase 活性，並且它與兩蛋白質之間的交互作用是相關聯的。另一個螢光探針 LY 被標定在 AtRGS1 的RGS 功能區塊的三個位置上，展示了在分子層次上兩蛋白質之間的交互作用機制。最後，根據螢光實驗資料，對於 AtGPA1 和 AtRGS1 之間交互作用的關係，我們提出一個兩階段交互作用的模型。

Heterotrimeric G-protein system serves many important roles in signal transduction and physiologies of mammalian and yeast. Upon ligand activation, G-protein coupled receptor (GPCR) could lead to a conformational change in Gα subunit and trigger the replacement of GDP for GTP in Gα. The Gα-GTP, activated form of Gα, dissociates from Gβγ subunit and GPCR, and initializes a series of signal cascading inside the cells. The signal then will be terminated through intrinsic GTPase enzyme activity of Gα which hydrolyzes the bound GTP into GDP and restores Gα to ground state. The GTPase activity of Gα can be further accelerated by a regulator of G-protein signaling (RGS) protein which interacts with Gα and turns off the Gα signaling state. Once GTP is hydrolyzed back to GDP, Gα will reassociate with Gβγ subunit again and ready for next stimulation. Similar to that of mammalian, Arabidopsis thaliana also harbors heterotrimeric G-protein system and each component of Arabidopsis heterotrimeric G-protein subunits involves many plant physiological functions, such as hormone sensing, cell proliferation and stress resistance. However, compared to the extensive studies on mammalian G proteins, research works on plant heterotrimeric G-protein system seldom focus on aspects other than their physiological functions. In this study, we use two different fluorescent probes, Lucifer yellow vinyl sulfone (LY) and BODIPYTR-GTP, to investigate the functions of Arabidopsis Gα subunit, AtGPA1, and AtRGS1, an Arabidopsis RGS protein, and interactions between them. By using BODIPYTR-GTP, we can real-time monitor protein interaction when RGS box domain of AtRGS1 specifically accelerates GTPase activity of AtGPA1. When another fluorescent probe, LY, was used to label at three different sites of RGS domain of AtRGS1, they can report the interaction and unveil a possible mechanism of these two proteins in molecular level. Finally, we propose a two-step interaction model for AtGPA1 and AtRGS1 based on these fluorescence data. In addition, we also screened crystallization conditions of AtGPA1 and RGS box domain of AtRGS1.