title: Localization of the RAR interaction domain of cellular retinoic acid binding protein-II.
abstract: The pleiotropic effects of retinoic acid (RA) in mammalian cells are mediated by two classes of proteins: the retinoic acid receptors (RAR), and cellular retinoic acid binding proteins (CRABP-I and CRABP-II). The high conservation across species and the differential expression patterns of the two CRABPs suggest that they serve distinct biological functions. We previously showed that CRABP-II, but not CRABP-I, delivers RA to RAR through direct protein-protein interactions between the binding protein and the receptor. "Channeling" of RA between CRABP-II and RAR markedly facilitates the formation of the holo-receptor and, as a consequence, enhances the transcriptional activity of RAR in cells. Here, we localize the region of CRABP-II that mediates the interactions of this protein with RAR. Comparison between the electrostatic surface potential of CRABP-I and II revealed the presence of a sole region displaying a dramatic potential change between the two isoforms. Examination of the underlying model revealed that the change stemmed from CRABP-I/CRABP-II substitution of three spatially aligned residues E75Q, K81P, and E102 K, located on a protrusion above the entrance to the ligand binding pocket of the protein. Substituting the corresponding CRABP-II residues onto CRABP-I conferred upon this protein the ability to channel RA to RAR and to enhance the transcriptional activity of RAR in cells. Conversely, converting these amino acid residues in CRABP-II to the homologous CRABP-I residues resulted in loss of the ability of CRABP-II to interact with RAR and to augment the receptor's activity. The data demonstrate that the surface region of CRABP-II containing residues Gln75, Pro81, and Lys102 is necessary and sufficient for mediating the interactions of this protein with RAR, facilitating the formation of the holo-receptor, and enhancing the transcriptional activity of RA.
authors.affiliation: Division of Nutritional Sciences, Cornell Theory Center, Cornell University, Ithaca, NY 14853, USA.
authors.forename: A
authors.initials: A
authors.lastname: Budhu
authors.forename: R
authors.initials: R
authors.lastname: Gillilan
authors.forename: N
authors.initials: N
authors.lastname: Noy
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D005453:Fluorescence; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D055672:Static Electricity; D015533:Transcriptional Activation; D014162:Transfection; D014508:Urea / Q000494:pharmacology
chemical_list: D018168:Receptors, Retinoic Acid; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; D014508:Urea