酸敏感G蛋白偶合受體功能上探討; Functional analysis of proton-sensing G-protein-coupled receptors

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題名:	酸敏感G蛋白偶合受體功能上探討;Functional analysis of proton-sensing G-protein-coupled receptors
作者:	蘇禹軒;Yeu-shiuan Su
貢獻者:	系統生物與生物資訊研究所
關鍵詞:	酸敏感G蛋白偶合受體;內在化;聚合體;oligomerization;FRET;internalization;g protein coupled receptors
日期:	2010-08-09
上傳時間:	2010-12-08 14:22:01 (UTC+8)
出版者:	國立中央大學
摘要:	G蛋白偶合受體 (GPCRs)屬於一種具有七個穿膜區域的受體，並傳遞細胞外來各種不同的刺激至細胞內產生反應。目前市面上約有五成的藥物是以GPCRs為目標但這些作為目標的GPCRs只佔了全部GPCR家族數量的百分之十，表示仍然有百分之九十的GPCRs是有潛力可以做為未來藥物的目標。而無法在細胞外獲得足量的GPCRs蛋白並獲得其結晶的結構，一直是研究GPCRs結構上很大的障礙。更複雜的是，G蛋白偶合受體被發現會以形成同源或是異源聚合體的方式來行使其生理功能。而這些同源和異源聚合體之間，或是單體和聚合體之間在受到相同配位體刺激後，卻會有不同的反應產生。這些聚合體的形成表示G蛋白偶合受體可以透過這種方式來接受不同配位體的刺激，產生不同的訊息傳遞路徑。有4個被認為是酸敏感的G蛋白偶合受體 (OGR1、GPR4、G2A、TDAG8) 被發現會在pH6.8至pH6.4時被完全的活化，且在生理pH值的恆定以及酸所引起的疼痛上有著重要的角色。在這些受體中，DNA序列比對的結果發現G2A與其他三個受體相似性較低，且相較於OGR1受體在被認有五個與接受氫離子相關的histidne胺基酸中，G2A有四個是被其他胺基酸所取代的。且以酸刺激G2A受體大量表現的細胞，意外地發現G2A受體並不會產生任何明顯的反應，但OGR1受體卻可以。因此，對於G2A受體是否會接受氫離子刺激或是可能與其他受體 (例如:OGR1)以形成異源聚合體的方式來行使其功能，至經仍然不清楚。本論文的目的是 (1) 於細菌中表現酸敏感GPCRs並純化供結構分析及 (2) 利用與配位體結合後產生內在化的現象來探討G2A受體是否會受酸所刺激並透過FRET 接受體光漂白的技術來觀察在酸的刺激下G2A與OGR1受體是否會以形成異源聚合體來產生反應。在這邊發現 (1) 酸敏感GPCRs無法順利的在細菌系統中表現；(2) G2A受體不會受酸所刺激並內在化到細胞質內；(3) G2A與OGR1受體會形成異源聚合體並對酸刺激產生反應。 G-protein-coupled receptors (GPCRs) that belong to seven transmembrane receptors, mediate a variety of extracellular signals to induce intracellular responses. Only 10% of GPCRs are targeted bt 50% of current marked drugs, emphasizing the potential of the remaining 90% of the GPCR superfamily for drug targets. The difficulties in large producing GPCRs in vitro and in generating crystal structures hinder structural studies of GPCRs. More complicatedly, GPCRs can form homo- or heteromers for function. The responses between homomers and heteromers or between oligomers and monomers are different while using the same agonist to stimulate. The oligomeric potential of GPCRs allows for more complex ligand-receptor relationships and signaling pathways. Four proton-sensing GPCRs (OGR1, GPR4, TDAG8, G2A) are identified to have fully activation at pH6.4~pH6.8 and important to pH homeostasis and acid-induced pain. Among these genes, primary sequence of G2A is less close to the others, and four of five critical histidine residues that are involved in pH sensing of OGR1 are replaced by other amino acids in G2A. Unexpectedly, G2A is the only proton-sensing GPCR that does not generate any significant responses after acid stimulation in cells overexpressing G2A gene, but did so in OGR1. Whether G2A does respond proton or forms heteromers with other family receptors (such as OGR1) to be functional, remains unclear. The objective of this thesis are (1) to purify proton-sensing GPCRs using bacteria expression system for structural analysis and (2) to explore whether G2A responds acid stimulation using ligand-mediated internalization technique and whether G2A form a functional heteromer with OGR1 to respond acid stimulation using FRET acceptor photobleaching technique. I have found that (1) bacteria expression system was not suitable for in vitro expression of proton-sensing GPCRs; (2) G2A did not internalize into the cytosol in response to acid stimulation, but OGR1 and TDAG8 did so; (3) G2A and OGR1 can form heteromers in resonse to acid stimulation.
顯示於類別:	[系統生物與生物資訊研究所] 博碩士論文

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