title: Purification and functional characterization of the ligand-binding domain from the retinoic acid receptor alpha: evidence that sulfhydryl groups are involved in ligand-receptor interactions.
abstract: 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)
authors.affiliation: CJF INSERM 92-03, Laboratoire de Biochimie Structurale, Faculté de Médecine, Lille, France.
authors.forename: N
authors.initials: N
authors.lastname: Dallery
authors.forename: B
authors.initials: B
authors.lastname: Sablonnière
authors.forename: I
authors.initials: I
authors.lastname: Grillier
authors.forename: P
authors.initials: P
authors.lastname: Formstecher
authors.forename: M
authors.initials: M
authors.lastname: Dautrevaux
mesh_terms: D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; D012176:Retinoids / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
chemical_list: D008024:Ligands; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; D012176:Retinoids; D013439:Sulfhydryl Reagents; D013917:Thrombin