阿拉伯芥與哺乳類動物之 G-protein 訊息系統相關蛋白質具有物種間交互作用

A Study on the Inter-species Interaction among G-protein Related Signaling Subunits from Arabidopsis thaliana and Mammals

黃柏勳(Po-Shiun Huang)

指導教授 ： 楊啟伸

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01200

摘要

異次三元體 Gα, Gβ, and Gγ 組成的 G 蛋白質為最常見的訊息傳遞分子，並且掌管許多重要的生理反應，是傳遞胞外訊息一個相當重要的媒介。其中以 Gα 次單元作為訊息啟動的樞紐，與 GTP 結合時轉為活化態並且引發下游訊息傳遞，直到 Gα 次單元本身的 GTPase 將 GTP 水解為 GDP 才終結反應。此外在G 蛋白質訊息途徑中，另一個關鍵性的蛋白質稱為 Regulator of G-protein signaling (RGS)，其在 G 蛋白質中扮演相當重要的角色，利用催化 Gα 次單元的 GTPase 活性以加速訊息傳遞的中止。過去在原生動物、真菌以及哺乳動物都相繼發現 RGS，直到最近才在阿拉伯芥中發現第一個植物 RGS (AtRGS1)，也是第一個被發現具有 N 端七個穿膜區結構的 RGS。本篇論文針對此 AtRGS1的 RGS domain 進行研究，利用動物的 Gα 次單元以及 RGS 進行物種間的交互作用，以螢光方法偵測 RGS 活性並佐以結構以及序列分析探討其物種間的差異。結果發現植物AtRGS1 也能催化動物 Gα 次單元 (Gαi1) 的 GTPase 活性，然而動物 RGS (RGS2, RGS4, and RGS9) 卻無法催化阿拉伯芥 Gα 次單元 (AtGPA1)。利用 Lucifer yellow 螢光標定的 RGS 即時偵測 AtRGS1 對於各種 Gα 次單元間的交互作用，結果顯示動物 Gαi1 及植物 AtGPA1 都能與 AtRGS1接觸，並且具有相似的親和性，因此進一步證實物種間的交互作用。綜合上述結果以及序列分析，植物 AtRGS1比較相似於動物 RGS4 所屬的R4 subfamily。雖然 AtRGS1 與動物 RGS 在保留性序列中大部分相似，但植物 AtRGS1 以及 AtGPA1 仍然具有其特殊性，因此可能是阻礙動物 RGS 與植物 AtGPA1交互作用的主要因素。

關鍵字

阿拉伯芥 ； G 蛋白質 ； 訊息傳遞 ； G 蛋白質訊息調節蛋白質 ； 螢光 ； 螢光標定 ； 物種間交互作用

並列摘要

Heterotrimeric G-protein, composed of Gα subunit and Gβγ dimer, is one of the most prevalent cellular signaling molecules and relays outside stimuli to mediate a variety of physiological responses. The G-protein signaling cascades are switched on via the GTP bound Gα subunit and then off due to the GTP hydrolysis that is catalyzed by the intrinsic GTPase of Gα subunit. Furthermore, this signaling pathway can be modulated by a group of proteins, regulator of G-protein signaling (RGS), which function as GTPase activating proteins (GAPs) to accelerate the GTPase activity of Gα proteins and then shorten the duration of signal transduction. Accordingly, RGS has been served as a negative regulator in G-protein signaling and its functional characteristic also makes RGS as a potential therapeutic target.
In mammalian, diverse RGS proteins form a RGS superfamily and are widely distributed in various tissues. However, in Arabidopsis, there is only one RGS protein (AtRGS1), the first RGS found to contain N-terminal seven-transmembrane domain. This study was designed to compare the similarity between the RGS domain of AtRGS1 and mammalian RGS proteins via fluorescent assay. The GAP activity for RGS proteins were conducted by the fluorescent GTP analogue, BODIPY TR-GTP, and the results exhibited that plant AtRGS1 can accelerate the GTPase activity of both cognate plant Gα (AtGPA1) and mammalian Gαi1, but this phenomenon was not consistent with mammalian RGS4, which only had GAP activity toward mammalian Gαi1 but not plant AtGPA1. These results were further confirmed by monitoring the interaction between Lucifer yellow-modified RGS and Gα proteins in real time. Moreover, the affinity assay also reported plant AtRGS1 had the similar affinity toward plant AtGPA1 and mammalian Gαi1, which further supported the existence of inter-species interaction. Finally, according to above results and coupling with structural and sequence analysis, it can be concluded that the plant AtRGS1 is more similar to the mammalian R4 subfamily RGS; however, AtRGS1 and AtGPA1 still contain some plant-specific residues to cause the barrier for inter-species interaction, thus blocking the interaction between mammalian RGS proteins and plant AtGPA1.

並列關鍵字

Arabidopsis thaliana ； G-protein ； signal transduction ； AtRGS1 ； AtGPA1 ； AtTHF1 ； RGS ； Gα ； fluorescence ； fluorescent labeling ； inter-species interaction

參考文獻

1. McCudden, C.R., Hains, M.D., Kimple, R.J., Siderovski, D.P. & Willard, F.S. G-protein signaling: back to the future. Cell Mol Life Sci 62, 551-77 (2005).

2. Cabrera-Vera, T.M. et al. Insights into G protein structure, function, and regulation. Endocr Rev 24, 765-81 (2003).

3. Oldham, W.M. & Hamm, H.E. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol 9, 60-71 (2008).

4. Bjarnadottir, T.K. et al. Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse. Genomics 88, 263-73 (2006).

5. Wettschureck, N. & Offermanns, S. Mammalian G proteins and their cell type specific functions. Physiol Rev 85, 1159-204 (2005).