A three-hybrid system for detecting small ligand-protein receptor interactions

被引:229
作者
Licitra, EJ
Liu, JO
机构
[1] MIT,CTR CANC RES,CAMBRIDGE,MA 02139
[2] MIT,DEPT BIOL,CAMBRIDGE,MA 02139
[3] MIT,DEPT CHEM,CAMBRIDGE,MA 02139
关键词
D O I
10.1073/pnas.93.23.12817
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo. This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined. The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506. Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and of FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone-FK506 heterodimer. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor-LexA DNA binding domain fusion protein in the presence of dexamethasone-FK506 heterodimer. We isolated overlapping clones of human FKBP12. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.
引用
收藏
页码:12817 / 12821
页数:5
相关论文
共 33 条
[1]  
Austin D J, 1994, Chem Biol, V1, P131, DOI 10.1016/1074-5521(94)90002-7
[2]  
AUSUBEL FM, 1995, CURRENT PROTOCOL MOL
[3]   Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins [J].
Belshaw, PJ ;
Ho, SN ;
Crabtree, GR ;
Schreiber, SL .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (10) :4604-4607
[4]  
CHAKRABORTI PK, 1991, J BIOL CHEM, V266, P22075
[5]  
COHEN JJ, 1989, ANTIINFLAMMATORY STE, P111
[6]   Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2 [J].
Colas, P ;
Cohen, B ;
Jessen, T ;
Grishina, I ;
McCoy, J ;
Brent, R .
NATURE, 1996, 380 (6574) :548-550
[7]   PEPTIDES ON PHAGE - A VAST LIBRARY OF PEPTIDES FOR IDENTIFYING LIGANDS [J].
CWIRLA, SE ;
PETERS, EA ;
BARRETT, RW ;
DOWER, WJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (16) :6378-6382
[8]   INTRACELLULAR LEUCINE ZIPPER INTERACTIONS SUGGEST C-MYC HETERO-OLIGOMERIZATION [J].
DANG, CV ;
BARRETT, J ;
VILLAGARCIA, M ;
RESAR, LMS ;
KATO, GJ ;
FEARON, ER .
MOLECULAR AND CELLULAR BIOLOGY, 1991, 11 (02) :954-962
[9]   RANDOM PEPTIDE LIBRARIES - A SOURCE OF SPECIFIC PROTEIN-BINDING MOLECULES [J].
DEVLIN, JJ ;
PANGANIBAN, LC ;
DEVLIN, PE .
SCIENCE, 1990, 249 (4967) :404-406
[10]  
ESTOJAK J, 1995, MOL CELL BIOL, V15, P5820