e kappa opioid receptor in this KCOS cell. Metabolic labeling of the rKOR prot

COS-1 細胞中大白鼠 Kappa 型類鴉片受體棕櫚酸修飾現象 之研究

Characterization of palmitoylation of rat kappa opioid recept or in COS-1 cells.

 棕櫚酸酯化作用 (palmitoylation) 是在蛋白質轉譯後 ， 蛋白質上的半胱胺酸 (cystei ne) 與棕櫚酸 (palmitic acid) 間形成硫酯鍵 (thioester bond) 的現象 。 這種修飾

作用已知存在於許多參與訊息傳遞的蛋白質上 ， 而且可能會影響到蛋白質的結構及功能

。 k 型類鴉片受體蛋白質 (KOR) 是屬於一種與 G 蛋白質偶合的細胞膜蛋白質 ， 根據 胺基酸序列的相似性比較 ，該蛋白質在靠近 COOH- 端的胺基酸 cysteine-340 被認為可 能是棕櫚酸酯化修飾發生的位置 。 在本篇論文中 ， 我們建立了一個經長期篩選而能大 量表現大白鼠 k 型類鴉片受體的 COS-1 細胞株 (KCOS) 。 我們以一具 k 型類鴉片受體 特異性的抗體 ， 於 KCOS 細胞中以螢光免疫染色確定該蛋白質的表現 。 並由西方雜交 法確定該 k 型類鴉片受體為一分子量為 43 kDa 以及一群約為 58~80 kDa 大小的膜蛋白質

。 此外 ， 我們亦利用聚合酵素連鎖增幅反應和北方雜交法加以確認類鴉片受體基因於 該 KCOS 細胞中的表現 。我們利用該細胞株進行類鴉片受體棕櫚酸酯化修飾的研究 。 經由 [3H] palmitate 代謝性標定實驗顯示該類鴉片受體蛋白可被 [3H] 標定 ， 表示此 受體蛋白質上可能有棕櫚酸酯化的修飾存在 。 而且經 [3H] 標定過的類鴉片受體蛋白以 1M 氫氧化銨處理後 ， 會使該代謝性標定現象消失 ， 表示此類鴉片受體蛋白的棕櫚酸 修飾作用是以硫酯鍵方式形成的 。

 Palmitoylation occurs posttranslationally on the cysteine residue through a th ioester linkage. It has been reported that the palmitoylation status of a memb rane-bound protein may influence the function and the structure of those prote ins. A potential palmitoylation site of the kappa opioid receptor which is bel onged to the family of transmembrane proteins coupling to G protein, has been recognized by sequence homology to be the cysteine340 residue. We have establi shed a stable tranfected COS-1 cells (KCOS) which can overexpress the rat kapp a opioid receptor (rKOR). We confirmed the expression of the rKOR on KCOS with a rKOR-specific antibody by immunofluorescence staining, and western blotting analysis. A protein with expected 43 kDa molecular weight and a group of pr

oteins with M.W. range from 58 to 80 kDa were detected in the membrane protein

s from KCOS cells. In addition, PCR amplification of the genomic DNA from the

KCOS cells and Northern blotting analysis further confirm the expression of th

e kappa opioid receptor in this KCOS cell. Metabolic labeling of the rKOR prot

ein by [3H]-palmitate in the KCOS cells showed that membrane proteins with the

same size of the rKOR was labeled with 3H, indicating the palmitate-modificat

ion. This metabolic labeling on the membrane protein of the KCOS cells was van

ished after pretreatment the protein with 1 M hydroxylamine, further showed th

e specific thioester linkage between palmitic acid and cysteine. .