Critical Role of Tyrosine 277 in the Ligand-Binding and Transactivating Properties of Retinoic Acid Receptor α

Abstract

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha 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 affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

“…[12][13][14][15][16][17][18][19][20] Because protein conformational changes affect their surface topology, amino acid residues involved in the structural changes can be identified from differential modification patterns. Information from surface mapping experiments is reliable, though, only if the structural integrity of a protein is preserved during the reaction.…”

Post-translational Modification of Nuclear Co-repressor Receptor-interacting Protein 140 by Acetylation

“…Post-translational modifications of transcription factors including nuclear receptors and their co-regulators have attracted much attention (21)(22)(23)(24)(25)(26). However, few studies have examined modifications of these proteins in vivo due to technical difficulties in expressing and purifying these nuclear proteins from eukaryotic cells.…”

Protein kinase C‐mediated phosphorylation of orphan nuclear receptor TR2: Effects on receptor stability and activity

“…In the recent years, post-translation modifications of nuclear receptors have drawn much attention [31][32][33][34]. However, few studies have taken steps to conduct vigorous studies such as using biochemical methods.…”

“…Factors affecting receptor's conformation, such as protein modification and interaction with coregulators, can play a crucial role in this regard. In the recent years, modulation of activities and stabilities of many nuclear receptors by specific post-translational modification including phosphorylation [31], acetylation [32], nitration [33], and glycosylation [34] have been suggested, primarily from molecular biological studies. Along this line, few studies have provided unambiguous, biochemical evidence for specific protein modifications on purified proteins.…”