Rat Cellular Retinol-binding Protein Research Articles

Two Homologous Rat Cellular Retinol-binding Proteins Differ in Local Conformational Flexibility

Cellular retinol-binding protein I (CRBP I) and cellular retinol-binding protein II (CRBP II) are closely homologous proteins that play distinct roles in the maintenance of vitamin A homeostasis. The solution structure and dynamics of CRBP I and CRBP II were compared by multidimensional NMR techniques. These studies indicated that differences in the mean backbone structures of CRBP I and CRBP II were localized primarily to the αII helix. Intraligand NOE cross-peaks were detected for the hydroxyl proton in the NOESY spectrum of CRBP I-bound retinol, but not for CRBP II-bound retinol, indicating that the conformational dynamics of retinol binding are different for these two proteins. As determined by Lipari–Szabo formalism, both the apo and holo forms of CRBP I and CRBP II are conformationally rigid on the pico- to nanosecond timescale. transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill -based 15N relaxation dispersion experiments at both 500 MHz and 600 MHz magnetic fields revealed that 84 and 62 residues for apo-CRBP I and II, respectively, showed detectable conformational exchange on a micro- to millisecond timescale, in contrast to three and seven residues for holo-CRBP I and II, respectively. Thus binding of retinol markedly reduced conformational flexibility in both CRBP I and CRBP II on the micro- to millisecond timescale. The 15N relaxation dispersion curves of apo-CRBP I and II were fit to a two-state conformational exchange model by a global iterative fitting process and by an individual (residue) fitting process. In the process of carrying out the global fit, more than half of the residue sites were eliminated. The individual chemical exchange rates k ex, and chemical shift differences, Δδ, were increased in the putative portal region (αII helix and βC-βD turn) of apo-CRBP II compared to apo-CRBP I. These differences in conformational flexibility likely contribute to differences in how CRBP I and CRBP II interact with ligands, membranes and retinoid metabolizing enzymes.

Structure and Backbone Dynamics of Apo- and Holo-cellular Retinol-binding Protein in Solution

Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo- and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo- and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligand-binding site. These comprise alpha-helix II, the subsequent linker to beta-strand B, the hairpin turn between beta-strands C and D, and the betaE-betaF turn. The internal backbone dynamics, obtained from 15N relaxation data (T1, T2, and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the betaC-betaD and betaE-betaF turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.

Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques.

Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction ( approximately 20%) retaining the initial methionine. By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E. coli grown in different media. Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used. In particular, this technique was applied to identify and quantify fatty acids bound to CRBP. Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein. Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression.

Transcriptional activation by hepatocyte nuclear factor-4 in a cell-free system derived from rat liver nuclei.

Hepatocyte nuclear factor-4 (HNF4) regulates gene expression by binding to direct repeat motifs of the RG(G/T)TCA sequence separated by one nucleotide (DR1). In this study we demonstrate that endogenous HNF4 present in rat liver nuclear extracts, as well as purified recombinant HNF4, activates transcription from naked DNA templates containing multiple copies of the DR1 element linked to the adenovirus major late promoter. Recombinant HNF4 also activates transcription from the rat cellular retinol binding protein II (CRBPII) promoter in vitro. The region between -105 and -63 bp of this promoter is essential for HNF-mediated transactivation. The addition of a peptide containing the LXXLL motif abolished HNF4-mediated transactivation in vitro suggesting that LXXLL-containing protein factor(s) are involved in HNF4-mediated transactivation in rat liver nuclear extracts. This is the first report on transactivation by HNF4 in a cell-free system derived from rat liver nuclei.

Binding of retinol induces changes in rat cellular retinol-binding protein II conformation and backbone dynamics

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(±0.09) Å. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the β-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the 15N T 1, T 2 and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T 2 derived apparent backbone exchange rate and amide 1H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.

Differential 9-cis-Retinoic Acid-dependent Transcriptional Activation by Murine Retinoid X Receptor α (RXRα) and RXRβ: ROLE OF CELL TYPE AND RXR DOMAINS

The 9-cis-retinoic acid (9cRA)-inducible enhancer of the rat cellular retinol-binding protein type II gene (CRBP II) was shown to be differentially regulated by the murine retinoid X receptor alpha (RXR alpha) as compared with RXR beta. Transient transfection assays performed in NIH 3T3 fibroblast cells demonstrated that RXR alpha yielded a high level of 9cRA-dependent transcription of a reporter gene linked to the CRBP II enhancer, when compared with RXR beta. This effect was cell type-dependent, since both receptors elicited comparable transcriptional activation of the same reporter in P19 embryonal carcinoma cells. To further explore the structural determinants responsible for the differences between these two receptors, a series of chimeric receptor constructs were made. Co-transfection assays utilizing these chimeras demonstrated that both the N terminus and the hinge region connecting the DNA binding domain with the ligand binding domain of RXR alpha were responsible for the high level of 9cRA-dependent transcription observed in NIH 3T3 cells, Furthermore, the hinge region of RXR alpha was shown to be necessary to repress, in the absence of hormone, the transcriptional activation function located in the N-terminal domain of RXR alpha. These results stress the importance of functional links between different RXR domains and suggest an RXR subtype and cell type-dependent specificity in the control of the 9cRA response.

Interactions with Retinol and Retinoids of Bovine Cellular Retinol-Binding Protein

The interactions with retinol and retinol analogs of bovine cellular retinol-binding protein (CRBP) have been investigated, by means of fluorescence titrations, to obtain more information on the structural features of retinoids that may be required for their interaction with the binding protein. An approximately stoichiometric binding of retinol to bovine CRBP (K′d∼2 nM) has been found in direct binding assays. Although retinal exhibited relatively high binding affinity to bovine CRBP (K′d∼ 30 nM), a large excess of the retinoid could not compete with retinol for the carrier protein. On the assumption that retinol and retinal interact with the same binding site, this result indicates that the above-mentioned apparent dissociation constant for retinol CRBP may be an overestimate and that its value may be as low as 0.1 nM. The finding of an exceedingly tight binding of retinol to CRBP provides further support for the possible role of CRBP-bound retinol, rather than its uncomplexed labile form, as substrate of enzymes involved in the metabolism of the vitamin. The results of these and previous studies indicate that CRBP is particularly sensitive to modifications of the retinol hydroxyl end group. Axerophthene, a retinol analog bearing a hydrogen atom in place of the hydroxyl end group, and β-ionone exhibit rather low binding affinities for CRBP (Kd∼ 0.2 μM and ∼ 4 μM, respectively), suggesting that the hydroxyl group and isoprene tail moieties contribute substantially to the retinol-binding affinity and specificity. These findings are consistent with the indications emerging from the three-dimensional structure determination of retinol CRBP [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1993) J. Mol. Biol. 230, 1225–1246]. Additionally, the bulky end groups of fenretinide and N-ethyl retinamide replacing the retinol hydroxyl group have been found to prevent retinoid binding to CRBP. The primary structure of bovine CRBP has been determined and is highly similar to the structures of both human and rat CRBP (97% and 95% identical, respectively).

Interactions with Retinol and Retinoids of Bovine Cellular Retinol‐Binding Protein

The interactions with retinol and retinol analogs of bovine cellular retinol-binding protein (CRBP) have been investigated, by means of fluorescence titrations, to obtain more information on the structural features of retinoid that may be required for their interaction with the binding protein. An approximately stoichiometric binding of retinol to bovine CRBP (K'd approximately 2 nM) has been found in direct binding assays. Although retinal exhibited relatively high binding affinity to bovine CRBP (K'd approximately 30 nM), a large excess of the retinoid could not compete with retinol for the carrier protein. On the assumption that retinol and retinal interact with the same binding site, this result indicates that the above-mentioned apparent dissociation constant for retinol.CRBP may be an overestimate and that its value may be as low as 0.1 nM. The finding of an exceedingly tight binding of retinol to CRBP provides further support for the possible role of CRBP-bound retinol, rather than its uncomplexed labile form, as substrate of enzymes involved in the metabolism of the vitamin. The results of these and previous studies indicate that CRBP is particularly sensitive to modifications of the retinol hydroxyl end group. Axerophthene, a retinol analog bearing a hydrogen atom in place of the hydroxyl end group, and beta-ionone exhibit rather low binding affinities for CRBP (K'd approximately 0.2 microM and approximately 4 microM, respectively), suggesting that the hydroxyl group and isoprene tail moieties contribute substantially to the retinol-binding affinity and specificity. These findings are consistent with the indications emerging from the three-dimensional structure determination of retinol.CRBP [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1993) J. Mol. Biol. 230, 1225-1246]. Additionally, the bulky end groups of fenretinide and N-ethyl retinamide replacing the retinol hydroxyl group have been found to prevent retinoid binding to CRBP. The primary structure of bovine CRBP has been determined and is highly similar to the structures of both human and rat CRBP (97% and 95% identical, respectively).

NMR studies of fluororetinol analogs complexed to two homologous rat cellular retinol-binding proteins

Comparative 19F-NMR studies of fluororetin ol analogs with rat cellular retinol binding protein II (CRBP II) and rat cellular retinol-binding protein (CRBP) were performed to probe differences in the binding interactions of these two homologous proteins. Line shape analyses of 19F-NMR spectra of ( E, E, Z, E)-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetren-1-ol (ligand 1), ( E, E, Z, E)-6-fluoro-9-(2,2′dimethyl-6-ethylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren-1-ol (ligand 2), ( E, Z, E, E)-5-fluoro-9-(2,2′-dimethyl-6-methylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren- l-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0–45°C, revealed that the 19F resonances corresponding to the bound ligand were in slow chemical exchange between two resonance frequencies. This was further supported by a 2D-NOESY exchange experiment. The k ex at 25°C was estimated from spectral simulation and fitting analyses to be 887 s ±1, 1010 s ±1and 771 s ±1 for CRBP II complexed 1, 2, and 3, respectively. In contrast, only a single absorption was observed for bound ligands complexed with rat CRBP over this temperature range, suggesting that the conformational dynamics of retinol binding are differeut for these two closely homologous proteins.

Constitutively active retinoid receptors exhibit interfamily and intrafamily promoter specificity.

Retinoid receptors are ligand activated transcription factors that regulate gene transcription through a complex network of interactions with members of the nuclear hormone receptor superfamily. Although ligand is required for trans-activation, addition of ligand to mammalian cells in vitro complicates the study of individual activated retinoid receptors. In order to circumvent this problem we have constructed a series of retinoid receptors which do not require ligand for trans-activation. This was accomplished by fusing the acidic activation domain of the herpes simplex viral protein VP16 to the carboxyl terminus of individual retinoid receptors. All of the chimeric receptors were found to exhibit constitutive trans-activation activity in CV-1 and P19 cells when cotransfected with a reporter that contained a trimerized retinoic acid receptor-beta 2 (RAR beta 2) retinoic acid response element. Further analysis conducted on reporters containing either the RAR beta 2 promoter or the rat cellular retinol binding protein II (rCRBPII) promoter showed that promoter specificity was well conserved between the chimeric receptors in the absence of exogenous retinoid and their ligand-induced native counterparts. Moreover, on the RAR beta 2 promoter reporter construct, the chimeric retinoid receptors displayed both cell type and inter- and intrafamily differences in trans-activation, whereas, trans-activation of the rCRBPII in the absence of exogenous ligand in CV-1 and P19 cells was found to be stimulated only by chimeric retinoid X receptor-alpha (RXR alpha). In P19 cells trans-activation of the rCRBPII promoter by RXR alpha v in the absence of exogenous ligand was inhibited by RAR alpha and the constitutive forms of RAR alpha, RAR beta, RAR gamma, RXR beta, and to a lesser extent RXR gamma.

The directly repeated RG(G/T)TCA motifs of the rat and mouse cellular retinol-binding protein II genes are promiscuous binding sites for RAR, RXR, HNF-4, and ARP-1 homo- and heterodimers.

We show here that the element which was previously characterized as a retinoid X receptor (RXR)-specific response element (RXRE) in the rat cellular retinol-binding protein II (CRBPII) gene is not conserved in the mouse gene. However, two conserved cis-acting elements (RE2 and RE3) located in the promoter region of the mouse and rat CRBPII genes mediate transactivation by retinoic acid receptors (RARs) and RXRs in transfected Cos-1, CV-1, and HeLa cells. The element RE3 which is the major retinoic acid (RA) response element also binds the transcription factors HNF-4 and ARP-1. HNF-4 constitutively activates the mouse CRBPII promoter, whereas ARP-1 represses the activation mediated by RARs, RXRs, and HNF-4. In contrast, RA has no effect on the activity of the mouse CRBPII promoter in the human colon carcinoma cell line CaCo-2 which constitutively expresses RAR alpha, RAR gamma, RXR alpha, HNF-4, and ARP-1, under conditions where the activity of the RAR beta 2 gene promoter is readily induced by RA. Our results suggest that the CRBPII gene may not be RA-inducible in tissues expressing HNF-4 and ARP-1, and that the RA inducibility of the CRBPII gene promoter observed in transfection experiments reflects the promiscuous binding of RARs/RXRs to HNF-4 and ARP-1 response elements.

Inter-tryptophan distances in rat cellular retinol binding protein II by solid-state NMR.

Structural constraints for the tryptophans in rat cellular retinol binding protein II (CRBP II) have been obtained by rotational-echo double-resonance (REDOR) solid-state NMR. CRBP II was labeled with L-[6-19F]tryptophan and L-[2-13C]tryptophan. The 13C-19F dipolar coupling was determined for various possible tryptophan geometries. The allowed distance between the closest two of the four tryptophans in CRBP II was obtained for each geometry. The minimum possible distance between these two tryptophans in CRBP II is 7 A, and the maximum possible distance is 11 A.

Differential binding of retinol analogs to two homologous cellular retinol-binding proteins.

A comparative study of the interactions of rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) with a number of synthetic phenyl-substituted analogs of all-trans-retinol was performed using fluorescence and nuclear magnetic resonance analysis. These studies indicate that CRBP II is more sensitive to modifications of the ring moiety than CRBP. Removal of the two methyl substituents on the ring which are ortho to the polyene chain abolishes binding to CRBP II. Conformational analysis of the ligands indicates that these two methyl groups influence the planarity of the ligand. The identification of monospecific ligands may prove useful for studying the physiological roles of these two proteins.

A retinoic acid response element from the rat CRBPI promoter is activated by an [formula omitted] heterodimer

The expression of the rat cellular retinol binding protein I (rCRBPI) can be upregulated in vivo by retinoic acid (RA). Here we have analyzed the rCRBPI promoter region and compared it to the corresponding mouse sequence. We find that the CRBPI 5′ flanking region has been highly conserved between rat and mouse, including a RA response element (RARE) ∼ 1 kb upstream of the start of transcription. The RARE is of the direct repeat type with a two nucleotide spacer. Like other direct repeat RAREs, this response element is activated by RARα and β but not by RARγ1. Furthermore, the rCRBPI-RARE is most effectively activated when both RAR and RXR are present. In addition RAR RXR heterodimers are required for efficient binding to the rCRBPI-RARE, while RARs or RXR alone do not interact effectively with this response element. The rCRBPI gene is therefore most likely activated in vitro by a rmRAR RXR heterodimer.

Heterodimeric receptor complexes determine 3,5,3'-triiodothyronine and retinoid signaling specificities.

Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) have been shown to interact with nuclear auxiliary proteins resulting in heteromeric complexes that bind strongly to their responsive elements. Recently the retinoid X receptors (RXRs) have been identified as one class of these nuclear proteins. RXRs strongly increase binding of TRs and RARs to a synthetic thyroid hormone (and retinoic acid) responsive element. Here results show that the binding of the heteromeric complexes to various natural response elements is highly specific and dictated by the partner of RXR in the complex. TR alpha and TR beta formed complexes with RXR alpha that strongly and selectively bound to natural thyroid hormone responsive elements, i.e. those from the rat alpha-myosin heavy chain gene and the rat malic enzyme gene. RXR alpha complexes with RAR alpha, RAR beta, and RAR gamma bound selectively to retinoic acid responsive elements from the human RAR beta 2 gene (hRAR beta 2), the gene of the rat cellular retinol binding protein I and the human apolipoprotein A1 gene. Under the conditions used here RXR alpha by itself did not bind to any of the responsive elements tested. Although TRs and RARs formed heterodimers with RXR in solution, these complexes were strongly stabilized by specific, high affinity response elements, but not by low affinity response elements. Transfection analyses showed strong synergism between receptors that formed effective heterodimers in transcriptional activation on several but not all response elements. Overall, these data demonstrate that RARs and TRs are unlikely to function as monomers or homodimers on the response elements investigated here and require RXRs or comparable proteins for effective response element activation.

Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants

Rat intestinal cellular retinol binding protein II (CRBP II) is an abundant 134-residue protein that binds all-trans-retinol which contains 4 tryptophans in positions 9, 89, 107, and 110. Our ability to express CRBP II in Escherichia coli and to construct individual tryptophan substitution mutants by site-directed mutagenesis has provided a useful model system for studying the fluorescence of a multi-tryptophan protein. Each of the four mutant proteins binds all-trans-retinol with high affinity, although their affinities are less than that of the wild-type protein. Steady-state and time-resolved fluorescence analyses of these proteins indicate that W107 is at the hydrophobic binding site, W110 is in a polar environment, and the remaining two tryptophans are in a hydrophobic environment. Time-resolved fluorescence study indicates that excited-state energy transfer occurs from the hydrophobic tryptophans to W110. The Stern-Volmer analysis with acrylamide of these proteins reveals that static quenching occurs in the W9F mutant protein while others do not. The fluorescence of rat intestinal fatty acid binding protein (I-FABP), a related protein of known X-ray structure, was also studied for comparison. The results of these findings, coupled with those derived from NMR studies and molecular graphics, suggest that CRBP II undergoes minor structural changes in all of the mutant proteins. Since these effects may be cumulative on the protein structure and function, any conclusions derived from higher mutants in this family of proteins must be treated with caution.

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.

Opposing Effects of Retinoic Acid and Dexamethasone on Cellular Retinol-Binding Protein Ribonucleic Acid Levels in the Rat*

Cellular retinol-binding protein (CRBP) is a potential mediator of vitamin A action. To determine whether retinoic acid and dexamethasone administration, alone and in combination, influence CRBP gene expression, adult female vitamin A-sufficient Sprague-Dawley rats randomly received 1) all-trans retinoic acid (100 micrograms) by intragastric intubation, 2) dexamethasone (2 micrograms/g BW) by ip injection, or 3) both all-trans retinoic acid and dexamethasone in the same doses. Control animals received either cottonseed oil by intragastric intubation or saline by ip injection. Six hours after treatment, lung and liver tissue were collected for Northern blot analysis with the radiolabeled cDNA specific for rat CRBP. Retinoic acid administration increased the amount of lung CRBP mRNA only, whereas dexamethasone decreased both lung and liver CRBP mRNA abundance. In animals treated with both retinoic acid and dexamethasone, CRBP mRNA abundance was also reduced. We conclude that CRBP gene expression can be modulated by both retinoic acid and dexamethasone in the vitamin A-sufficient animal. In the whole animal, our findings indicate that dexamethasone not only represses CRBP gene expression, but also opposes the effect of retinoic acid.

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.

Site-directed mutagenesis of rat cellular retinol-binding protein: Alteration in binding specificity resulting from mutation of glutamine 108 to arginine

Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein. A rat cDNA clone of CRBP was expressed in Escherichia coli. In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins. Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function. Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R). Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex. The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity. In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol. Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.