第五亞型蕈毒乙醯膽鹼受體在酵母菌細胞膜之功能性表現

Abstract

如同哺乳動物細胞，酵母菌的表面受體也可經由G—蛋白將訊息傳遞到細胞內。前人的論文指出大鼠的Gsα基因放入scg1基因缺失的酵母菌突變株內可補回部分酵母菌G蛋白複合體之生理功能。本論文試圖將大鼠之受體蛋白放到酵母菌中表現以觀察下列現象(1)外來受體蛋白在酵母菌中是否還能維持原來的空間結構(2)外來受體蛋白是否與酵母菌本身的G-蛋白結合。 本論文所構築的質體是利用酵母菌費洛蒙基因的?動子與分泌性前導勝?序列來表現大鼠的第五亞型簟毒乙醯膽鹼受體(M5)，再經由轉形(transformation)至酵母菌後得到以下的結果(1)M5可以成功的表現，並被運移至細胞膜上(2)當酵母菌細胞被費洛蒙刺激後會造成費洛蒙受體被磷酸化，並導至M5與ligand的專一性結合能力喪失(3)尚未發現M5能與酵母菌本身的G-蛋白結合之証據。

As mammalian cells, yeast cells also contain surface receptors systems coupling to G protein signal transduction pathways. It was previously shown that expression of rat Gsα in yeast cells will complement the growth and morphological defects of an scg1 null mutant. To facilitate structure-functional analysis of G protein coupled receptor, the rat fifth muscarinic acetylcholine receptor (M5) has been expressed in S. cerevisiae. This was achieved by placing a truncated M5 gene in the yeast pYaM5 vector under the control of the yeast α-factor promoter and secretory leader sequence. Results deduced from saturative binding experiments indicated that M5 expressed on yeast plasma membrane displayed characteristic affinities and specifity to its ligand. When the phosphorylation was triggered by addition of pheromone induction, the M5 on this conditioned cells lost its binding activity to ligand. The coupling of M5 to G proteins of pheromone signal transduction pathways is not detected.