Localization of the RAR interaction domain of cellular retinoic acid binding protein-II.

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. Copyright 2001 Academic Press.