The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.     

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

Comparative 19F-NMR studies of fluororetinol 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-methylcyclohexenyl)-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+ ++-1-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0-45 degrees 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 kex at 25 degrees C was estimated from spectral simulation and fitting analyses to be 887 s-1, 1010 s-1 and 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 different for these two closely homologous proteins.     

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In response to estrogen the rat cervical epithelium undergoes squamous metaplastic changes, progressing from a resting state through a proliferating, secretory stage and finally to a cornified stage before sloughing or being reabsorbed. The transition from a secretory to a cornified epithelium is preceded by a dramatic reduction in the expression of the cellular retinol binding protein (CRBP). The associations among retinoids (retinol and retinoic acid), CRBP expression, and estrogen-induced keratinocyte differentiation were explored in cultured cervical epithelial cells. Retinoids supported proliferation of cervical epithelial cells expressing basal keratins. Alone, estrogen had no effect on proliferation and enhanced expression of keratins characteristic of stratified cervical epithelial cells. When added together, estrogen prevented retinoid effects on proliferation, whereas retinoids prevented the estrogen-enhanced expression of differentiation-associated cytokeratins. When CRBP expression was repressed by elevating intracellular cyclic AMP levels, the ability of retinol, but not retinoic acid, to block estrogen-induced changes in keratin expression was severely compromised. These results support a critical role for CRBP in cervical cell responsiveness to circulating retinoids (primarily retinol). We hypothesize that retinol inhibits estrogen-induced keratinization of the cervical epithelium, and the drop in CRBP level results in transient vitamin A deficiency within cervical epithelial cells, permitting the orderly transition from the secretory to the cornified stage.     

Retinoic acid stimulates early cellular proliferation in the adapting remnant rat small intestine after partial resection

Following loss of small bowel surface area, the remnant intestine undergoes a remarkable adaptive response. To define more fully the underlying molecular mechanisms, we have identified genes that are specifically induced in the adapting remnant after partial small bowel resection. Several of these, including cellular retinol binding protein II (CRBP II) and apolipoprotein (apo) AI, participate in vitamin A and lipid trafficking. The CRBP II and apo A-I promoters contain response elements for the nuclear retinoid X receptor RXR-alpha. It is well established that vitamin A is essential for normal cell growth, differentiation and maintenance of epithelial tissues and that CRBP II functions to facilitate intestinal vitamin A absorption and metabolism. On the basis of these considerations, changes in CRBP II and apo A-I mRNA levels could reflect a role for retinoids in modulating the intestinal adaptive response. To explore this hypothesis, we used a rat resection model of intestinal adaptation to examine the temporal patterns of CRBP II, apo A-I and RXR-alpha expression postresection. CRBP II and apo A-I mRNA levels were increased in the remnant intestine in distinct temporal patterns, whereas RXR-alpha expression was unchanged. To address directly the effects of vitamin A in adaptation, retinoic acid or vehicle was administered intravenously to rats immediately after 70% small bowel resection. Compared with vehicle, all-trans-retinoic acid significantly stimulated crypt cell proliferation in the adapting remnant intestine by 6 h after surgery. These data suggest that retinoic acid acts to modulate intestinal proliferation in the adapting small intestine after loss of functional small bowel surface area.     

Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis

Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.     

Retinol transport into the cell nucleus in vitro

The retinol interaction with intact cellular nuclei, nuclear envelope and chromatin was investigated. We have shown that the Cellular Retinol-Binding Protein (CRBP) plays a very important role in such interaction. Retinol can specifically interacts with nuclei, nuclear envelope and chromatin only when it presents as a complex with CRBP. The obtained data allowed us to suggest that the process of retinol delivery to hypothetical nuclear receptors include of at least the following stages: 1) specific binding with nuclear envelope; 2) penetration into nuclei; 3) specific binding with chromatin receptors. CRBP is a necessary component at all this stages. Also we show that CRBP has not a species or tissue specificity.     

The regulation of retinoic acid formation

Rat liver microsomal as well as cytosolic retinol dehydrogenases from cellular retinal-binding protein (CRBP)-retinal from CRBP-retinol. A cytosolic dehydrogenase transforms retinal into retinoic acid. Apo-CRBP and ethanol inhibit the cytosolic but not the microsomal retinol dehydrogenase, the latter being the primary enzyme in the rate-limiting step in retinoic acid synthesis.     

Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation

The expression of cellular retinoic-acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP), as well as their relationship to retinoic acid (RA) synthesis and degradation were examined in the developing mouse cerebellum and choroid plexus of the fourth ventricle. The choroid plexus, which expresses the RA-synthesizing retinaldehyde dehydrogenase RALDH-2, is likely to represent a diffusion source of RA for the closely apposed cerebellum, regulating its development. We found CRBP to be expressed in the choroid plexus and, in an in-vitro assay, addition of recombinant CRBP to RALDH-2 increased RA synthesis from retinaldehyde, with the amount of increase depending on the CRBP/retinaldehyde ratio. A technique that characterizes RA-binding proteins according to their isoelectric point showed both CRABP I and CRABP II to be present in the cerebellum and P19 cells, and only CRABP II to be present in the choroid plexus. With this technique, CRABP I could also be detected in the HL60 cell line. In addition to the two known acidic RA-binding proteins CRABP I and II, the cerebellum expressed a third RA-binding protein distinguishable by its neutral isoelectric point; the same binding protein was also detected in the olfactory bulb, kidney and testes. We used the RA-binding technique to follow the rate of elimination of bound RA from the cerebellum. A systemic injection of 0.3 micromols RA into postnatal day-1 mice was almost completely removed after 8 hours. These results suggest mechanisms by which the retinoid-binding protein may regulate the equilibrium of RA synthesis and catabolism in the cerebellum and choroid plexus.     

Substrate specificity and kinetic mechanism of the insect sulfotransferase, retinol dehydratase

Spodoptera frugiperda retinol dehydratase catalyzes the conversion of retinol to the retro-retinoid anhydroretinol. It shares sequence homology with the family of mammalian cytosolic sulfotransferases and provides the first link between sulfotransferases and retinol metabolism. In this study the enzymatic properties of retinol dehydratase were examined using bacterially expressed protein. We show that retinol dehydratase can catalyze the transfer of the sulfonate moiety to small phenolic compounds and exhibits many functional similarities to the mammalian cytosolic sulfotransferases. The bisubstrate reaction that it catalyzes between retinol and the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate seems to involve ternary complex formation and to proceed via a Random Bi Bi mechanism. In addition to the low nanomolar Km value for free retinol, retinol dehydratase is strongly inhibited by retinol metabolites, suggesting a preference for retinoids. Conversely, a number of tested mammalian cytosolic sulfotransferases do not utilize retinol, indicating that retinol is not a general substrate for sulfotransferases.     

N-(4-hydroxyphenyl)-retinamide increases lecithin:retinol acyltransferase activity in rat liver

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02-2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 3H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.     

Intracellular events in the "selective" transport of lipoprotein-derived cholesteryl esters

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the "selective" uptake and intracellular itinerary of lipoprotein-derived cholesteryl esters. Bt2cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cell's plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.     

Urinary retinol excretion and kidney function in children with shigellosis

BACKGROUND: Acute infections, including diarrhea, are associated with an increased risk of vitamin A deficiency. Urinary retinol excretion during such infections may contribute to this risk. The mechanism accounting for urinary retinol loss has not been clearly defined. OBJECTIVE: This study attempted to determine whether urinary retinol loss in children with acute infection is associated with impaired kidney function, particularly impaired tubular protein reabsorption. DESIGN: Urinary retinol excretion and kidney function were examined in 66 hospitalized children 5 mo to 5 y of age with acute Shigella dysentery. RESULTS: Urinary retinol loss occurred in 59% of children and was substantial (>0.1 micromol/d) in 8% of them. Children with more severe disease excreted higher concentrations of urinary retinol; those with a body temperature > or =40 degrees C excreted a mean of 0.10 +/- 0.18 micromol/d compared with 0.005 +/- 0.008 micromol/d for other children (P < 0.0001). Children with more severe disease also had impaired tubular reabsorption of low-molecular-weight proteins beta2-microglobulin and retinol binding protein (RBP)], although other measures of tubular and glomerular function were not similarly impaired. In multiple regression analysis, severity of disease indicators were the best predictors of tubular reabsorption of beta2-microglobulin (R2 = 0.53) whereas tubular reabsorption of beta2-microglobulin and RBP were found to be the best predictors of urinary retinol loss (R2 = 0.69). CONCLUSIONS: A significant amount of retinol was excreted in the urine in children with shigellosis: 8% excreted >0.10 micromol/d (15% of the daily metabolic requirement). Impaired tubular reabsorption of low-molecular-weight proteins, such as RBP transporting retinol, appeared to be the cause of this urinary retinol loss.     

Physical determinants of intermembrane protein transfer

Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.     

Reduced lecithin:retinol acyl transferase activity in cultured squamous cell carcinoma lines results in increased substrate-driven retinoic acid synthesis

The uptake and metabolism of retinol was compared in squamous cell carcinoma lines, SCC12b and SCC13, and in normal human keratinocytes (NHK). Long chain fatty acid esters of retinol and 3,4-didehydroretinol were the predominant metabolites formed in both cell types. Lesser amounts of unesterified retinol, 3,4-didehydroretinol, and their respective active acid forms were also observed. Despite a qualitatively similar retinoid composition, there were significant quantitative differences between cell types. Most notable was that SCC formed only about one-fourth the retinoid ester as did normal cells. In parallel with this, unesterified retinol and retinoic acid concentrations in SCC were significantly elevated over those in normal cells. This altered pattern of retinoid metabolites in SCC was found to be due to very low lecithin:retinol acyltransferase (LRAT) activity. SCC exhibited less than one-tenth the LRAT activity of normal cells. Acyl-coenzyme A:retinol acyltransferase (ARAT) and retinyl ester hydrolase activities were not different between cell types. Challenging cells with increasing medium retinol concentrations resulted in dose-dependent increases in retinol and retinoic acid within SCC. In contrast, retinol and retinoic acid concentrations in similarly challenged normal cells remained relatively low across a wide retinol concentration range. This was accomplished by the storage of retinol, via LRAT activity, as retinyl ester. Consistent with increased substrate-driven retinoic acid synthesis in SCC, the expression of transglutaminase 1 was suppressed to a greater extent in the SCCs than in NHK, when cells were exposed to equivalent medium concentrations of retinol. The data demonstrate a central role of LRAT in regulating retinoic acid synthesis via its capacity to modulate cellular levels of substrate retinol.     

Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [3H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP-TTR complex was nearly twofold greater than that from RBP alone. The uptake of [3H]retinol from protein-bound retinol was inhibited by an excess of either retinol-RBP or retinol-RBP-TTR complex. Moreover, retinol uptake through the RBP-TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

The myristoyl moiety of myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein is embedded in the membrane

Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.     

Effects of fasting and 3,3',4,4',5,5'-hexabromobiphenyl on plasma transport of thyroxine and retinol: fasting reverses elevation of retinol

Male Wistar rats were injected ip with 0 or 20 mg/kg 3,3',4,4',5,5'-hexabromobiphenyl and blood samples were collected 1, 3, 6, 7, and 8 d later. At 8 d after the injection, serum retinol was increased 30% and serum thyroxine was decreased 26% relative to control values. These effects were apparently unrelated to transthyretin in that the biphenyl did not alter the proportion of thyroxine binding in vitro to the prealbumin fraction of serum proteins. Separate groups of control and HBBP-injected rats did not receive food on d 7 (i.e., 24-h fast) and d 8 after injection (i.e., 48-h fast). Fasting decreased the serum retinol and thyroxine concentrations as well as the proportion of thyroxine binding in vitro to the prealbumin fraction of serum. The decreases in retinol and thyroxine concentrations associated with fasting are therefore ascribed to a decrease in the concentration of transthyretin in circulation.