Critical Role of Tyrosine 277 in the Ligand-Binding and Transactivating Properties of Retinoic Acid Receptor
详细信息 查看全文
- 作者：Sandrine Mailfait ; Denise Belaiche ; Mostafa Kouach ; Nathalie Dallery ; Philippe Chavatte ; Pierre Formstecher ; and Bernard Sablonniè ; re
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 7, 2000
- 年：2000
- 卷：39
- 期：9
- 页码：2183 - 2192
- 全文大小：512K
- 年卷期：v.39,no.9(March 7, 2000)
- ISSN：1520-4995
文摘
Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RAR
, 
, and 
is sensitive to nitrationwith tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that areimportant for RA binding, we carried out chemical modification experiments with purified RAR ligand-binding domain (RAR-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemicallymodified peptides containing each of the three Tyr residues present in the RAR-LBD sequence werethen analyzed and identified by high-performance liquid chromatography coupled to electrospray ionizationmass spectrometry (HPLC/ESI-MS). We found that RA binding to RAR-LBD protected Tyr277-containingpeptides from nitration. Protection of Tyr277 could result either from direct masking by the bound ligandor from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residueswas further documented by site directed mutagenesis using three site-specific RAR mutants: Y208A,Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinityand transactivation activity for RA. Whereas mutation of Tyr277 into alanine had a variable effect ondifferent agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivationactivity. This later effect was also observed with mutation of Tyr277 into phenylalanine. It is unlikely thatmajor conformational changes are responsible for the lower affinity of RA binding and RA-dependenttransactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limitedproteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightlydifferent from that obtained with the wild-type protein. These data could suggest that Tyr277 play a criticalrole in the ligand-induced conformational changes required for the activation of RAR.
, , and is sensitive to nitrationwith tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that areimportant for RA binding, we carried out chemical modification experiments with purified RAR ligand-binding domain (RAR-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemicallymodified peptides containing each of the three Tyr residues present in the RAR-LBD sequence werethen analyzed and identified by high-performance liquid chromatography coupled to electrospray ionizationmass spectrometry (HPLC/ESI-MS). We found that RA binding to RAR-LBD protected Tyr277-containingpeptides from nitration. Protection of Tyr277 could result either from direct masking by the bound ligandor from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residueswas further documented by site directed mutagenesis using three site-specific RAR mutants: Y208A,Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinityand transactivation activity for RA. Whereas mutation of Tyr277 into alanine had a variable effect ondifferent agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivationactivity. This later effect was also observed with mutation of Tyr277 into phenylalanine. It is unlikely thatmajor conformational changes are responsible for the lower affinity of RA binding and RA-dependenttransactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limitedproteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightlydifferent from that obtained with the wild-type protein. These data could suggest that Tyr277 play a criticalrole in the ligand-induced conformational changes required for the activation of RAR.