Distinct Roles for Cellular Retinoic Acid-binding Proteins I and II in Regulating Signaling by Retinoic Acid

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). Here we show that expression of CRABP-II, but not CRABP-I, markedly enhanced RAR-mediated transcriptional activation of a reporter gene in COS-7 cells. The equilibrium dissociation constants of complexes of CRABP-I or CRABP-II with RA were found to differ by 2-fold. It is thus unlikely that the distinct effects of the two proteins on transactivation stem from differential ligand-binding affinities. The mechanisms by which RA transfers from the CRABPs to RAR were thus investigated directly. The rate constant for movement of RA from CRABP-II, but not from CRABP-I, to RAR strongly depended on the concentration of the acceptor. The data suggest that transfer of RA from CRABP-I to RAR involves dissociation of the ligand from the binding protein, followed by association with the receptor. In contrast, movement of RA from CRABP-II to the receptor is facilitated by a mechanism that involves direct interactions between CRABP-II and RAR. These findings reveal a striking functional difference between CRABP-I and CRABP-II, and point at a novel mechanism by which the transcriptional activity of RA can be regulated by CRABP-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). Here we show that expression of CRABP-II, but not CRABP-I, markedly enhanced RAR-mediated transcriptional activation of a reporter gene in COS-7 cells. The equilibrium dissociation constants of complexes of CRABP-I or CRABP-II with RA were found to differ by 2-fold. It is thus unlikely that the distinct effects of the two proteins on transactivation stem from differential ligand-binding affinities. The mechanisms by which RA transfers from the CRABPs to RAR were thus investigated directly. The rate constant for movement of RA from CRABP-II, but not from CRABP-I, to RAR strongly depended on the concentration of the acceptor. The data suggest that transfer of RA from CRABP-I to RAR involves dissociation of the ligand from the binding protein, followed by association with the receptor. In contrast, movement of RA from CRABP-II to the receptor is facilitated by a mechanism that involves direct interactions between CRABP-II and RAR. These findings reveal a striking functional difference between CRABP-I and CRABP-II, and point at a novel mechanism by which the transcriptional activity of RA can be regulated by CRABP-II. retinoic acid retinoic acid receptor cellular retinoic acid-binding protein retinoid X receptor dioleylphosphatidychloline Retinoic acid (RA)1 is an important regulator of cell growth and differentiation both in fetal and in adult tissues. Two classes of proteins are involved in mediating the multiple biological activities of RA. One of these, the retinoic acid receptors (RARs), encompasses ligand-inducible transcription factors that belong to the superfamily of nuclear hormone receptors and that are specifically activated by RA. RARs associate with the retinoid X receptor (RXR) to form RAR-RXR heterodimers, which regulate transcription following binding at the promoter regions of various target genes and activation by their cognate ligands (1Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2622) Google Scholar). Another class of intra-cellular proteins that bind RA with a high affinity comprises two homologous proteins, cellular RA-binding proteins I and II (CRABP-I and CRABP-II). CRABPs are found in all vertebrates and are highly conserved across species (2Ong D.E. Newcomer M.E. Chytil F. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, NY1994: 288-317Google Scholar). The two CRABP isoforms display different patterns of expression across cells and developmental stages. In the adult, CRABP-I is expressed almost ubiquitously, whereas CRABP-II is only expressed in skin (2Ong D.E. Newcomer M.E. Chytil F. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, NY1994: 288-317Google Scholar), uterus, ovary (3Zheng W.L. Ong D.E. Biol. Reprod. 1998; 58: 963-970Crossref PubMed Scopus (47) Google Scholar, 4Wardlaw S. Bucco R.A. Zheng W.L. Ong D.E. Biol. Reprod. 1997; 56: 125-132Crossref PubMed Scopus (61) Google Scholar), and in the choroid plexus (5Yamamoto M. Drager U.C. Ong D.E. McCaffery P. Eur. J. Biochem. 1998; 257: 344-350Crossref PubMed Scopus (56) Google Scholar). Both CRABPs are widely expressed in the embryo, although they do not usually co-exist in the same cells (6Maden M. Blomhoff R. Vitamin A in Health and Disease. Marcel Dekker, NY1994: 289-322Google Scholar). The distinct patterns of expression of CRABP-I and II suggest that they serve different functions in the biology of RA, or, perhaps, that they allow for accommodating different requirements for RA in different tissues. Although the genes for both CRABP isoforms from various species have been cloned and characterized, neither the exact functions of these proteins nor the distinct roles of the two isoforms are completely understood at present. Interestingly, despite the high conservation of CRABPs, mice in which these genes have been disrupted appear essentially normal (7Gorry P. Lufkin T. Dierich A. Rochtte-Egly C. Decimo D. Dolle P. Mark M. Durand B. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9032-9036Crossref PubMed Scopus (122) Google Scholar). It is usually proposed that CRABPs serve to solubilize and protect their ligand in cytosol and that they transport RA between different cellular compartments (2Ong D.E. Newcomer M.E. Chytil F. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, NY1994: 288-317Google Scholar). It was suggested, for example, that CRABPs act to deliver their ligand to the nucleus (8Takase S. Ong D.E. Chytil F. Arch. Biochem. Biophys. 1986; 247: 328-334Crossref PubMed Scopus (161) Google Scholar). Indeed, it was recently demonstrated that both CRABP isoforms are present not only in cytosol but also in the nuclei of cells (9Gaub M-P. Lutz Y. Ghyselinck N.B. Scheuer I. Pfister V. Chambon P. Rochette-Egly C. J. Histochem. Cytochem. 1998; 46: 1103-1111Crossref PubMed Scopus (58) Google Scholar). It was also suggested that CRABP-I regulates the metabolic fate of its ligand by directly affecting the activities of RA-metabolizing enzymes. It was reported that the rate of degradation of RA in F9 teratocarcinoma cells increases upon elevation of the expression level of CRABP-I (10Boylan J.F. Gudas L.J. J. Biol. Chem. 1992; 267: 21486-21491Abstract Full Text PDF PubMed Google Scholar). It was shown further that the sensitivity of F9 cells to RA-induced differentiation is inversely correlated to the cellular level of CRABP-I (11Boylan J.F. Gudas L.J. J. Cell Biol. 1991; 12: 965-979Crossref Scopus (310) Google Scholar). Hence, it is currently believed that CRABP-I moderates cellular response to RA by facilitating catabolism and/or by sequestering RA, rendering it unavailable to nuclear receptors. Little information is available regarding the specific biological role of CRABP-II. Here, the kinetic patterns that govern the process by which CRABPs deliver their ligand to RAR were examined and correlated with their effects on RAR-mediated transcriptional activation. The observations indicate that CRABP-II, but not CRABP-I, directly interacts with RAR and that these protein-protein interactions markedly facilitate the formation of the RAR·RA complex. hRARα Lacking the terminal A/B domain (RARαΔAB) was obtained by overexpression in Escherichia coli and purified as described previously (13Kersten S. Kelleher D. Chambon P. Gronemeyer H. Noy N. Proc. Natl. Acad. Sci. U. S. A. 1995; 96: 10014-10017Google Scholar). This protein displays ligand-binding, DNA-binding, and dimerization properties that are identical to those of the full-length protein (14Chen Z.-P. Iyer J. Bourguet W. Held P. Mioskowski C. Lebeau L. Noy N. Chambon P. Gronemeyer H. J. Mol. Biol. 1998; 275: 55-65Crossref PubMed Scopus (61) Google Scholar). Bacterial expression vectors for bCRABP-I and bCRABP-II (in pT7 vector) were provided by David Ong (Vanderbilt University). These proteins were expressed and purified as described (15Jamison R.S. Newcomer M.E. Ong D.E. Biochemistry. 1994; 33: 2873-2879Crossref PubMed Scopus (58) Google Scholar). COS-7 cells were transfected with pSG5 vector containing either CRABP-I or CRABP-II (2 μg), together with DR-5-tk-CAT reporter plasmid (1 μg, see Ref. 23Daniels C. Noy N. Zakim D. Biochemistry. 1985; 24: 3286-3292Crossref PubMed Scopus (102) Google Scholar for details) and pCH110 (0.5 μg). Assays were carried out as described previously (14Chen Z.-P. Iyer J. Bourguet W. Held P. Mioskowski C. Lebeau L. Noy N. Chambon P. Gronemeyer H. J. Mol. Biol. 1998; 275: 55-65Crossref PubMed Scopus (61) Google Scholar). were carried out as described previously (16Kersten S. Dawson M.I. Lewis B.A. Noy N. Biochemistry. 1996; 34: 3816-3824Crossref Scopus (45) Google Scholar) and followed either by monitoring the ligand-induced decrease of the fluorescence of the protein (λex = 280 nm, λem = 340 nm) (17Cogan U. Kopelman M. Mokady S. Shinitzky M. Eur. J. Biochem. 1976; 65: 71-78Crossref PubMed Scopus (362) Google Scholar) or the increase in fluorescence of RA upon binding to CRABP (λex = 360 nm, λem = 470 nm) (18Fiorella P.D. Giguere V. Napoli J.L. J. Biol. Chem. 1993; 268: 21545-21552Abstract Full Text PDF PubMed Google Scholar). Titration curves were corrected (24Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 112-150Google Scholar) and data fitted to a binding equation (19Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (75) Google Scholar). Unilamellar vesicles of DOPC (Avanti Polar Lipids) were prepared as described previously (20Noy N. Xu Z.-J. Biochemistry. 1990; 29: 3878-3883Crossref PubMed Scopus (109) Google Scholar). Holo-CRABP-I or -II was mixed with vesicles, and RA transfer was monitored by following either the fluorescence of the protein or the fluorescence of RA. Data were fitted to a first order reaction. Equimolar solutions of CRABP and RA were mixed using a stopped-flow apparatus (HiTech, Salisbury, UK), and complex formation was monitored by following the time-dependent increase in RA fluorescence. Data were fitted to a second order equation. holo-CRABP was mixed with RAR at varying RAR/CRABP molar ratios in the range of 5–25. Transfer was monitored by the time-dependent decrease in the fluorescence of RA. Data were fitted to a first order equation. To investigate whether either CRABP-I or CRABP-II affects the transcriptional activity of RA, transactivation assays were carried out. A CAT reporter construct containing the response element DR-5, which specifically binds RAR-RXR heterodimers (21Mangelsdorf D.J. Umesono K. Evans M.R. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, NY1994: 319-349Google Scholar), was co-transfected into COS-7 cells together with expression vectors for either CRABP-I or CRABP-II, and the ability of RA to activate transcription of the reporter gene was studied (Fig. 1). Addition of RA induced expression of the reporter in a dose-dependent fashion (Fig. 1, open bars). Overexpression of CRABP-I had little effect on the RA-induced activation (Fig. 1, gray bars). In contrast, expression of CRABP-II markedly stimulated transactivation by RA (Fig. 1, hatched bars) in this system. To examine whether functional differences between CRABP-I and CRABP-II may stem from differences in their RA binding affinity (19Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (75) Google Scholar), Kd values characterizing the association of RA with CRABP-I and CRABP-II were measured. To this end, the kinetic parameters of the dissociation and the association of complexes of RA with the binding proteins (koff and kon, respectively) were measured. The respective Kdvalues were obtained by using the relationship Kd =koff/kon. To withdraw RA from CRABP, unilamellar vesicles of DOPC, serving as a “hydrophobic sink,” were used (22Chen Y. Houghton L.A. Brenna J.T. Noy N. J. Biol. Chem. 1996; 271: 20507-20515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). CRABP was pre-complexed with RA, mixed with the vesicles, and movement of the ligand from the protein to the vesicles was followed. RA fluoresces when bound to CRABP-I or II, although it is not fluorescent when associated with lipid vesicles (18Fiorella P.D. Giguere V. Napoli J.L. J. Biol. Chem. 1993; 268: 21545-21552Abstract Full Text PDF PubMed Google Scholar). RA transfer from CRABP to vesicles could thus be followed by monitoring the time-dependent decrease in the fluorescent of the ligand. In this assay, because the amount of vesicles used was sufficient to draw >95% of the ligand from the protein, the rate constant of the observed reaction directly reflects the rate constant for dissociation of RA from the protein (koff) (16Kersten S. Dawson M.I. Lewis B.A. Noy N. Biochemistry. 1996; 34: 3816-3824Crossref Scopus (45) Google Scholar, 20Noy N. Xu Z.-J. Biochemistry. 1990; 29: 3878-3883Crossref PubMed Scopus (109) Google Scholar). Representative traces showing transfer of RA from CRABP-I or CRABP-II to vesicles are shown in Fig.2, a and b, respectively, and the derived rate constants are listed in TableI. The rate of association of the CRABP·RA complexes was examined by mixing equimolar concentrations of RA and protein and following the time-dependent enhancement of the fluorescence of the ligand upon binding (Fig. 2, c and d). These data were analyzed as described previously (20Noy N. Xu Z.-J. Biochemistry. 1990; 29: 3878-3883Crossref PubMed Scopus (109) Google Scholar) to yield kon (Table I). The data indicated that kon for the two proteins are similar and close to the diffusion limit and that koff is 2-fold lower for CRABP-I versus CRABP-II, leading to a 2-fold higher ligand-binding affinity of the former.Table IParameters characterizing the interactions of RA with CRABPs1-aMean ± S.E., n = 5–9.Proteinkofft1/2 offkonKdmin−1minm−1min−1nmCRABP-I0.22 ± 0.013.23.55 ± 0.38 × 1090.06CRABP-II0.42 ± 0.051.73.08 ± 0.08 × 1090.131-a Mean ± S.E., n = 5–9. Open table in a new tab It is difficult to see how a 2-fold difference in the Kd of CRABP-I and II may account for their distinct effects on the transcriptional activity of RAR. We thus wondered whether the functional differences between the two proteins may stem from differences in the mechanisms by which they deliver their ligand to RAR. Theoretically, transfer of a ligand from a donor to an acceptor protein may occur by one of two possible mechanisms. One pathway involves initial dissociation of the ligand from the donor into the aqueous phase, followed by association with the acceptor. In this case, the rate-limiting step for the transfer reaction will be the dissociation of the donor-ligand complex, and the rate constant of the reaction will be independent of the nature or the concentration of the acceptor (23Daniels C. Noy N. Zakim D. Biochemistry. 1985; 24: 3286-3292Crossref PubMed Scopus (102) Google Scholar). In a second scenario, the ligand will move from the donor to the acceptor by “channeling,” i.e. by a process that involves direct protein-protein interactions and that bypasses the aqueous phase. In this case, the rate of the reaction will be limited by the frequency of productive collisions between the donor and the acceptor and will become faster as the acceptor/donor ratio is increased. Hence, the mechanism by which CRABP “delivers” RA to RAR can be delineated by examining the dependence of the rate constants of ligand transfer between the two proteins on the concentration of the acceptor (RAR). As RA is an efficient fluorophore when bound to either CRABP but not when associated with RAR, transfer could be followed by the time-dependent decrease in RA fluorescence upon mixing of holo-CRABP with apo-RAR. The rate constant for movement of the RA from CRABP-I to RAR was independent of the concentration of the acceptor (Fig. 3 a), indicating that movement of RA from CRABP-I to RAR requires prior dissociation of the ligand from CRABP-I. To verify the accuracy of the fluorescence measurements, the rate of transfer was also measured by monitoring movement of 3H-RA from CRABP-I to RAR. Following mixing, separation of the two proteins at different time points was affected by addition of Ni2+ chelating beads which bind the his-tagged RAR but not CRABP. Mixtures were centrifuged, and the remaining CRABP-bound RA was measured by counting. The rate constant thus obtained was essentially identical to that extracted from the fluorescence assays (data not shown). In with CRABP-I, the first order rate constant for movement of RA from CRABP-II to RAR (Fig. 3 strongly depended on the concentration of the a increase in the concentration of RAR facilitated that rate of transfer of RA from CRABP-II to RAR by This strongly that movement of RA from CRABP-II to RAR is mediated by direct protein-protein interactions between the two the data reveal that CRABP-I as a for RA which binds and its ligand in response to in equilibrium CRABP-II RA to RAR by between the two These observations show further that the interactions between CRABP-II and RAR in of the formation of the complex and may be the for the effect of CRABP-II on the RAR-mediated To further the interactions that transfer of RA between CRABP-II and RAR, we to formation of a complex between the two The following were (13Kersten S. Kelleher D. Chambon P. Gronemeyer H. Noy N. Proc. Natl. Acad. Sci. U. S. A. 1995; 96: 10014-10017Google Scholar), fluorescence (24Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 112-150Google D. Noy N. Biochemistry. 1998; PubMed Scopus Google Scholar), to examine whether cognate may the complex D. Noy N. Biochemistry. 1998; PubMed Scopus Google Scholar). We could not complex formation using of these It thus that the interactions of complex is an with a Two classes of proteins are believed to be involved in the transcriptional activities of the retinoid nuclear receptors RAR and and the cellular RA-binding proteins CRABP-I and CRABP-II. The mechanisms of of retinoid receptors have become understood in despite the striking level of conservation of CRABP across which suggest that they roles in RA and although the different expression of the two CRABP isoforms suggest that they different roles in RA neither the exact functions of these proteins nor the nature of their distinct roles are at present. The was thus to investigate possible functional differences between CRABP-I and CRABP-II. The data in that expression of CRABP-II markedly the RA-induced transcriptional activity of RAR and in contrast, CRABP-I effect on this Hence, the two proteins display functional It was previously shown that expression of CRABP-I in F9 teratocarcinoma cells the transcriptional activity of RAR, and it was suggested that CRABP-I RA to that its degradation (10Boylan J.F. Gudas L.J. J. Biol. Chem. 1992; 267: 21486-21491Abstract Full Text PDF PubMed Google Scholar, J.F. Gudas L.J. J. Cell Biol. 1991; 12: 965-979Crossref Scopus (310) Google Scholar). In contrast, the data in 1, in with carried out using cells P. Biochemistry. 1996; PubMed Scopus Google Scholar), show that expression of CRABP-I had little effect on RA-induced transactivation in COS-7 cells. It thus that the effect of CRABP-I on the transcriptional activity of RAR on the cell It is to for example, that CRABP-I the transcriptional activity of RA only in cells that RA-metabolizing that are its direct were demonstrated to in F9 cells (11Boylan J.F. Gudas L.J. J. Cell Biol. 1991; 12: 965-979Crossref Scopus (310) Google Scholar). To of RA in either COS-7 or not been studied in The Kd values of complexes of RA with CRABP-I and -II were found to differ by only 2-fold (Table that it is unlikely that the functional differences between the two proteins stem from differential ligand-binding affinities. We thus out to examine whether CRABP-I and -II deliver their ligand to RAR by different mechanisms. Theoretically, are two possible by which a ligand can move from a donor to an acceptor Transfer may by dissociation of the ligand from the donor to the aqueous phase, followed by association with the acceptor. In this the rate of transfer will be limited by the rate of dissociation of the donor-ligand complex. the ligand may transfer from the donor to the acceptor by a process that is mediated by direct protein-protein In this case, the rate of transfer will on the of productive collisions between the two proteins and will be faster as the acceptor/donor ratio is increased. of the kinetic patterns of the movement of RA between the binding proteins and RAR (Fig. demonstrated that transfer of RA from CRABP-I to RAR is of a process that requires prior dissociation of the complex This is also by the observations that the rate constant for dissociation of RA from CRABP-I was similar of whether the acceptor was RAR or lipid vesicles (Table I). In with CRABP-I, the of movement of RA from CRABP-II to RAR was of a process that is mediated by direct interactions between the two proteins the data that these interactions in a of the of RA to the receptor. Hence, CRABP-II the transcriptional activity of RAR by directly with the facilitating the formation of the RAR·RA complex. Interestingly, it was recently reported that expression of CRABP-II transcriptional activation by RA in cell Y. S. R. 1997; Google Scholar) and that expression of a CRABP-II construct in cells these cells to of 1998; Google Scholar). for the mechanism that may these observations were in these but the of the present suggest that these effects were mediated by the of the formation of It is also that it been reported that the expression of CRABP-II is in cells that of RA (5Yamamoto M. Drager U.C. Ong D.E. McCaffery P. Eur. J. Biochem. 1998; 257: 344-350Crossref PubMed Scopus (56) Google Scholar, W.L. Bucco R.A. Ong D.E. 1996; PubMed Scopus Google Scholar, R.A. Zheng W.L. J.T. E. Ong D.E. Biochemistry. 1997; PubMed Scopus Google Scholar). These observations can be in of the present to that for RA both an increase in RA and an of CRABP-II, for of RA to RAR. to a complex between CRABP-II and RAR despite of multiple It thus that the interactions between the two proteins are in nature and that the complex is a interactions that serve to ligands between proteins have been previously for for the pathway J. J. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). In these similar to the present it was not possible to a complex between the proteins involved although it been that direct protein-protein interactions occur and an important role in this metabolic To the of the present the first for of a ligand between two proteins for movement of a a metabolic We for of of transfer of 3H-RA between CRABP-I and RAR. We are to David for expression vectors for CRABP-I and CRABP-II, and to Gronemeyer and for for CRABP-I, CRABP-II, and RAR and for the reporter