臺灣博碩士論文加值系統

腺苷酸2A亞型受體(A2A adenosine receptor,簡稱為A2AR)屬於G蛋白耦合
受體，A2AR在所有腺苷酸受體中擁有最長的細胞質羧基端(cytoplasmic carboxyl terminus)。之前的研究顯示A2AR與許多生理或病理反應有關：包括血管增生，傷口癒合，抗發炎反應及腦部缺血。A2AR也參與於許多神經退化性疾病，如帕金森氏症(Parkinson’s diseases)及亨丁頓舞蹈症(Huntington’s disease; HD)的病程與治療，而尋找C端交互作用蛋白的研究的開端就是為了瞭解這個特長的C端是否有助於A2AR的功能。本篇論文中發現了一個新型的C端交互作用蛋白，Gas-2 like 2 (G2L2)。A2AR與G2L2的結合可以用GST融合蛋白沈澱法, 共免疫沈澱法, 細胞染色(ICC), 及螢光共振能量轉移(FRET)等實驗方法証實，而A2AR的活化對G2L2的結合與否沒有明顯的影響。雖然G2L2的氨基酸序列在不同物種間的差異不大，但是到目前為止，對於G2L2的功能仍舊十分不清楚。G2L2蛋白在大腦各區中都有表現，在細胞膜上的raft特化微結構區中也被發現會與A2AR及活化型G蛋白(Gαs)共同存在。在亨丁頓舞蹈症的動物模型中G2L2的蛋白表現量明顯的增加，之前研究也顯示此動物模型之A2AR的訊息傳導會被異常地放大。於是，本篇論文證實了在細胞內大量表現G2L2可以增加A2AR所誘導生成的cAMP，而這個功能依賴著G2L2與A2AR的 C端的結合。我們的研究團隊之前已經報導過活化A2AR有助於減輕或減緩亨丁頓舞蹈症的病癥與病程。因此，G2L2可藉由結合A2AR而活化A2AR訊息傳導的這個功能是否與減輕或減緩亨丁頓舞蹈症的病癥與病程有關，在未來便可用幾項指標測試：包括AMP-activated protein kinase (AMPK)的磷酸化程度，以及胞內蛋白被泛素化(ubiqutination)或形成不正常蛋白聚集的程度。此外，在活化A2AR的過程中，G2L2的表現不但增加活化態Gαs (Gαs-GTP)的量，Gαs-GTP可被偵測到的時間點也提早了一點。G2L2與Gαs的結合在本篇研究中也已經共免疫沈澱及融合蛋白沈澱法證實。而G2L2偏好結合未活化態的Gαs (Gαs-GDP)的現象正說明了G2L2可藉由招募G蛋白三聚合體到A2AR的附近以利A2AR訊息有效率地傳導。在不同細胞株的測試中，我也陸續發現了G2L2可能具備其他的功能。舉例而言，在ST14A及NSC34細胞株中，當細胞在養分不足(serum starvation)或遭遇氧化壓力(oxidative stress)時，G2L2的表現量會上升。在PC12及N2A細胞株進行分化時，G2L2似乎也參與了神經纖維生長及延伸的過程。綜合以上論述，G2L2被新發現為一個可與A2AR結合的蛋白，而G2L2可經由與A2AR交互作用進而控制並微調由Gαs媒介而生成的cAMP含量。希望將來G2L2在體內做的進一步的鑑定，對於針對A2AR在神經性疾病及創傷的製藥領域中可以有長足的幫助。

The A2A adenosine receptor (A2AR) is a G-protein-coupled receptor that contains a long cytoplasmic carboxyl terminus (A2AR-C). Previous studies have demonstrated that the A2AR is involved in many physiological and pathological conditions including angiogenesis, wound healing, anti-inflammation responses, cerebral ischemia and several neurodegenerative diseases such as Parkinson’s diseases and Huntington’s diseases (HD). To examine whether this unique long C-terminus contributes to the function of A2AR, I set out to search for proteins interacting with the C-terminus of A2AR. In the thesis, I report that Gas-2 like 2 (G2L2) is a new interacting partner of A2AR-C. The interaction between A2AR and G2L2 was verified by GST pull-down, co-immunoprecipitation (co-IP), immunocytochemical (ICC) staining, and fluorescence resonance energy transfer (FRET). Receptor activation by CGS, however, caused little effect on the G2L2-A2AR interaction. Amino acid sequence of G2L2 is conserved among species, yet its function is largely unknown. G2L2 proteins were found in the brain. In the brain, G2L2 also coexisted in the raft microdomain of the membrane where A2AR and Gαs resided in. In the striatum of a mouse model of HD, expression of G2L2 was markedly enhanced, which was associated with an abnormal amplification of the cAMP signal evoked by A2AR. Expression of G2L2 increased the intracellular cAMP content evoked by A2AR in an A2AR-C-dependent manner. Binding of G2L2 to A2AR therefore might be beneficial for HD and could be assessed by indexes including the level of phosphorylated AMP-activated protein kinase (AMPK), ubiquitinylated proteins, and mutant huntingtin protein aggregates in the future. During receptor activation, the cellular content of activated Gαs (Gαs-GTP) was increased faster in the presence of G2L2. Immunoprecipitation and pull-down assays demonstrated that G2L2 selectively bound to Gαs. Binding of the inactive form of Gαs to G2L2 facilitated the recruitment of the trimeric G protein complex to the proximal position of A2AR for efficient activation. I also found a few additional functions of G2L2 in several cell lines tested. For example, G2L2 was upregulated when ST14A and NSC34 cells were under serum starvation or oxidative stress. G2L2 was also involved in the neuritogenesis of different cell lines (i.e., PC12 and N2A cells). Collectively, G2L2 is a new effector that controls the action of A2AR by modulating its ability to regulate the Gαs-mediated cAMP contents. Further characterization of the role of G2L2 in vivo would greatly advance our current understanding of A2AR as a drug target for neuronal diseases and traumas.

Contents
1.中文摘要 1
2. Abstract 3
3. Introduction 5
3.1 The A2A adenosine receptor (A2AR) 5
3.2 The regulator of G protein signaling (RGS) 5
3.3 The GPCR-Interacting Proteins (GIPs) 6
3.4 GIPs of A2AR 8
3.5 Spectraplakins 8
3.6 Growth-arrest-specific 2-like protein 2 (G2L2) 10
3.7 Aims of the study 11
4. Materials and methods 12
5. Results 24
5.1. A novel Gαs-binding protein, G2L2, facilitates the signaling of A2AR. 24
5.1.1. Search for novel interacting protein(s) of the C-terminus of A2AR. 24
5.1.2. G2L2 enhances A2AR-mediated cAMP signaling via direct binding. 28
5.1.3. G2L2 regulates A2AR signaling by binding to Gs. 29
5.2. Characterizations and potential functions of G2L2. 32
5.2.1. Distribution of G2L2. 32
5.2.2. G2L2 may be upregulated in R6/2 mice. 34
5.2.3. Direct binding of G2L2 and A2AR may ameliorate the disease progression of HD. 35
5.2.4. G2L2 contains a regulator of G-protein signaling (RGS)-like domain. 36
5.2.5. G2L2 is upregulated upon stress. 39
5.2.6. G2L2 may enhance the retinoid acid (RA)-induced neurite outgrowth in Neuro-2A (N2A) cells. 40
5.2.7. Knockdown and knockout of G2L2 40
6. Discussion 44
7. Future perspectives 53
8. Reference 56
9. Figures 69
10. Tables 105

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