The cellular transport of retinoic acid (RA) and 9-cis-RA is believed to be mediated by their cellular RA-binding protein, CRABP. The biological activity of these retinoids in the control of cellular differentiation and growth is mediated by their nuclear receptors, RARs (alpha, beta, and gamma) for RA and RXRs (alpha, beta, and gamma) for 9-cis-RA. The mechanism by which the nuclear receptors accept their ligands from the cytosol and are activated is not known, and forms the central theme of this proposal. The efficacy of RAs complexed with CRABP as compared with free RAs in the ligand-nuclear receptor interactions will be determined by using purified receptor protein preparations. The specific role of RA- response elements (REs) in enhancing/stabilizing the RA binding to the receptors, and the role of CRABP/RA in producing such effects will be elucidated by using a gel mobility shift assay. Also to be established will be, by the use of intact nuclei, the mode of transfer of RAs into the nuclei for their interactions with RAR/RXR. Experiments will be devised to utilize expression plasmids harboring full-length cDNA for CRABP, RAR and RXR to elucidate the specific role of CRABP in the binding of the ligands to the transiently expressed receptors in CV-1 cells. A CRABP- mediated functional role of RAR/RXR will be delineated by using a cis- trans cotransfection assay which would utilize cDNA expression vectors for the nuclear receptors and RARE/RXRE-chloramphenicol acetyl transferase. By extending these efforts, identification of possible activator/antagonist synthetic retinoids (from a rationally selected group of retinoids) that would alter the transcriptional activation of the receptors will also be accomplished. The unifying hypothesis to be tested is that CRABP plays a role not only in the transport of RAs to their nuclear receptors but that it also has a functional role.
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