|Table of Contents|

Specific labeling of the cell membrane-bound estrogen receptor(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2014 02
Research Field:
Publishing date:


Specific labeling of the cell membrane-bound estrogen receptor
YUAN Yun LIN Zhiwei GUO Jiajia WANG Fei YUAN Xiaohong
1College of Life Sciences, Southwest University of Science and Technology, Mianyang 621000, China 2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 3Chengdu University of Technology, Chengdu 610059, China
estrogen receptor phosphopantetheinyl transferase labeling coenzyme A
Q291 : Q78

Cell membrane-bound estrogen receptors (cmERs) mediate the non-genomic effects of estrogen, which is involved in the occurrence of breast cancer and many other estrogen-related diseases. However, the mechanism study is hindered by the lack of a specific labeling method which can efficiently differentiate between cmERs and cytoplasmic ERs. The purpose of this study was to develop the specific labeling of the cmER. We expressed and purified glutathione S-transferase fusion proteins which contained three different specific peptides A1, S6 and ybbR. Then we compared the labeling specificity and efficiency of the proteins by two different phosphopantetheinyl transferase (PPTase): AcpS and Sfp. A1 peptide, which was shown with higher labeling efficiency, was constructed into the C-terminus of the estrogen receptor α (ERα-A1) and then the ERα-A1 expression, its genomic and nongenomic function and labeling of biotin by AcpS were measured in cells. The results showed that coupling of A1 tag did not affect the ER protein expression or its activity. AcpS could specifically label biotin to cmERα-A1with the coenzyme A-biotin as a substrate. These results provide a new method for the study of cmER-mediated estrogen nongenomic signal transduction.


1 Dagmar DH, Stephan S, Dietrich K. Inhibition of human: cytochrome P450 aromatase activity by butyltins [J]. Steroids, 2001, 66: 763-769
2 梅春霞, 张吉强. 雌激素受体[J]. 生命化学, 2010, 30 (4): 590-594 [Mei CX, Zhang JQ. Estrogen receptor [J]. Chem Life, 2010, 30 (4): 590-594]
3 Johnsson K, George N. Methods for protein labeling based on acyl carrier protein: United States, US 7, 666, 612 B2 [P]. Feb. 23, 2010
4 Kropf M, Rey G, Glauser L, Kulangara K, Johnsson K, Hirling H. Submit-specific surface mobility of differentially labeled AMPA receptor subunits [J]. Eur J Cell Biol, 2008, 87: 763-778
5 Zhou Z, Cironi P, Lin AJ, Xu YQ, Hrvatin S, Golan DE, Silver PA, Walsh CT, Yin J. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases [J]. Acs Chem Biol, 2007, 2 (5): 337-346
6 Viver-Pol L, George N, Krumm H, Johnsson K, Johnsson N. Multicolor imaging of cell surface proteins [J]. J Am Chem Soc, 2005, 127 (37): 12770-12771
7 Yin J, Lin AJ, Buckett PD, Wessling-Resnick M, Golan DE, Walsh CT. Single-cell FRET imaging of transferring receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling [J]. Chem Biol, 2005, 12: 999-1006
8 Yin J, Straight PD, Mcloughlin SM, Zhou Z, Lin AJ, Golan DE, Kelleher NL, Kolter R, Walsh CT. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase [J]. Proc Nat Acad Sci USA, 2005, 102 (44): 15815-15820
9 Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand Binding [J]. FASEB J, 1995, 9 (5): 404-410
10 Kuroki Y, Fukushima K, Kanda Y, Mizuno K, Watanabe Y. Putative membrane-bound estrogen receptors possibly stimulate mitogen-activated protein kinase in the rat hippocampus [J]. Eur J Pharmacol, 2000, 400: 205-209
11 Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR. Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling [J]. Proc Nat Acad Sci USA, 2000, 97 (11): 5930-5935
12 Wu XQ, Oppermann U. High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system [J]. J Chromatogr B, 2003, 786 (1): 177-185
13 Liang SP. A Course of Biochemistry and Molecular Biology Experiment [M]. Beijing: Higher Eduction Press, 2003
14 Yin J, Lin AJ, Golan DE, Walsh CT. Site-specific protein labeling by Sfp phosphopantetheinyl transferase [J]. Nat Protocols, 2006, 1: 280-285
15 Hall JM, McDonnell DP. The estrogen receptor β-isoform (ERβ) of the human estrogen receptor modulates ERα transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens [J]. Endocrinol, 1999, 140 (12): 5566-5578
16 杨丽娟, 黄君梅, 王飞. 何首乌、骨碎补和淫羊藿的植物雌激素作用研究[J]. 中药与临床, 2012, 3 (3): 37-40 [Yang LJ, Huang JM, Wang F. Evaluation of the phytoestrogenic activity of Polygonum multiforum, Drynaria fortune and Epimedium brevicornu [J]. Pharm Clin Chin Mat Med, 2012, 3 (3): 37-40]
17 Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK. Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance [J]. Clin Cancer Res, 2004, 10: 331S-3316S
18 魏明宝, 崔长征, 方呈祥, 张甲耀, 王海, 王亚芬. 铜绿假单胞菌增强型绿色荧光蛋白标记的研究[J]. 应用与环境生物学报, 2005, 11 (1): 74-77 [Wei MB, Cui CZ, Fang CX, Zhang JY, Wang H, Wang YF. EGFP marker of Pseudomonas aeruginosa [J]. Chin J Appl Environ Biol, 2005, 11 (1): 74-77]
19 陈蕾蕾, 陈军浩, 孙雪梅, 施广飞. 荧光染料CFSE作为细胞标记的特性研究[J]. 细胞与分子免疫学杂志, 2004, 20 (2): 140-141 [Chen LL, Chen JH, Sun XM, Shi GF. The characteristics of fluorescent dye CFSE as cell labeled research [J]. Chin J cell Mol Immunol, 2004, 20 (2): 140-141]
20 Wade CB, Robinson S, Shapiro RA, Dorsa DM. Estrogen receptor (ERα) and ERβ exhibit unique pharmacologic properties when coupled to activation of the mitogen-activa protein kinase pathway [J]. Endocrinology, 2001, 142 (6): 2337-2342
21 Qiu J, Bosch MA, Tobias SC, Grandy DK, Scanlan TS, Ronnekleiv OK, Kelly MJ. Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C [J]. J Neurosci, 2003, 23 (29): 9529-9540
22 Brailoiu E, Dun SL, Brailoiu GC, Mizuo K, Sklar LA, Oprea TI, Prossnitz ER, Dun NJ. Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system [J]. J Endocrinol, 2007, 193: 311-321
23 Song RX, Barnes CJ, Zhang ZG, Bao YD, Kumar R, Santen RJ. The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. Proc Nat Acad Sci USA, 2004, 101: 2706-2801


Last Update: 2014-05-04