The pGEX-2T expression vector was used to produce the ligand-binding domain from the human retinoic acid receptor alpha (hRAR alpha LBD) in Escherichia coli. The resulting fusion protein, containing the glutathione S-transferase separated from the truncated receptor (hRAR alpha 186-462) by a thrombin cleavage site, was purified with use of affinity chromatography on immobilized glutathione. A 90% homogeneity was obtained, with a specific activity of 100 pmol/mg and an overall 10% yield. Following purification and thrombin cleavage, a predominant monomeric (stokes radius = 2.3 nm, molecular mass of 32 kDa) [3H]retinoic acid hRAR alpha LBD complex was characterized by high-performance size-exclusion chromatography. The purified hRAR alpha LBD bound retinoic acid with an apparent Kd of 9 nM, a value close to the Kd of the full-length hRAR alpha expressed in COS cells. Kinetic studies at 0 degrees C demonstrate that the association of [3H]retinoic acid and [3H]CD367, a synthetic retinoid, to the overexpressed receptor was extremely rapid (complete in less than 3 min), whereas their dissociation from the receptor was slower, with half-lives of about 40 min at 0 degrees C. Experiments performed at various subzero temperatures allowed a more accurate assay of the association rate constant and indicate that the entropy of activation (delta Sa) is positive, which is characteristic of hydrophobic interactions. The ligand-binding activity was markedly decreased by pretreatment with various sulfhydryl modifying agents. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) appeared to be the most potent, whereas iodoacetamide was the least active. Furthermore, a series of N-alkylmaleimides was shown to inactivate the recombinant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

No abstract