Retinol-binding Protein Receptor Research Articles

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.

Comparison of the Metabolism of Retinol Delivered to Human Keratinocytes either Bound to Serum Retinol-Binding Protein or Added Directly to the Culture Medium

Retinol (vitamin A) circulates in the blood bound to retinol-binding protein (RBP), which is thought to be responsible for the delivery of the vitamin to target cells, including the basal cells of the skin (keratinocytes). The process by which keratinocytes acquire retinol from RBP remains controversial. A mechanism for retinol delivery to keratinocytes involving cell surface RBP receptors has been proposed, while other studies support an RBP receptor-independent process. To further explore retinol uptake we have used a model system of human foreskin keratinocytes cultured in serum-free media to compare the metabolism of [3H]retinol delivered to the cells either bound to RBP or added directly to the culture medium. The majority of the cell-associated radioactivity found in keratinocytes incubated for 0.5 to 24 h with either free or RBP bound [3H]retinol was present as [3H]retinyl ester irrespective of the mode of delivery. In keratinocytes incubated for 24 h with [3H]retinol added directly to the culture medium or bound to RBP, [3H]retinyl ester comprised 76 and 80%, respectively, of the total cell-associated radioactivity. Also, the relative cellular levels of the different retinyl esters species synthesized by the keratinocytes were the same whether the [3H]retinol was delivered free or bound to RBP. Finally, the kinetics of loss (turnover) of cell-associated [3H]retinol and [3H]retinyl esters from keratinocytes prelabeled with [3H]retinol delivered free or bound to RBP was the same. Overall, this study demonstrates that the rate and extent of retinol esterification by keratinocytes and the types of esters synthesized are the same whether the vitamin is delivered to the cells free or bound to RBP and argues against RBP receptor-mediated delivery of retinol to specific sites on the plasma membrane that influence overall retinol metabolism.

Identification of a mechanism to localize generation of retinoic acid in rat embryos

Vitamin A (retinol) is essential for normal mammalian development. However, its biological activity depends upon its conversion to retinoic acid (RA), a local mediator of cellular proliferation and differentiation. Previous studies have shown that embryonic RA is found specifically in tissues known to depend upon vitamin A for normal development and that its production follows uptake of maternal retinol. The aim of this study was to identify the mechanism for tissue-specific generation of RA in developing rat embryos. Here we show immunohistochemical localization of the retinol binding protein receptor, cellular retinol binding protein, retinol dehydrogenase and retinal dehydrogenase in rat embryos (presomitic to the 25–30 somite pair stage). These proteins are proposed to be responsible for cellular uptake of retinol, its intracellular transport and its conversion to RA. Thus, they potentially constitute the entire metabolic pathway from vitamin A to RA. All four proteins were detected specifically in tissues that are known to depend upon vitamin A for normal development including the yolk sac, heart, gut, notochord, somites, sensory placodes and the limb. Furthermore, our previous studies have demonstrated that uptake of retinol into the yolk sac depends upon a retinol binding protein receptor. Here we provide evidence that this mechanism functions also in the heart. Colocalization of cellular retinol binding protein, retinol and retinal dehydrogenase with the retinol binding protein receptor in tissues dependent upon vitamin A for normal development suggests that coordinate functioning of these proteins is responsible for cellular uptake of circulating retinol and its metabolism to RA. This is the first evidence of a tissue-specific mechanism for generation of RA from its precursor retinol in the developing embryo.

A retinol-binding protein receptor-mediated mechanism for uptake of vitamin A to postimplantation rat embryos.

The major form of dietary vitamin A in blood is retinol bound to retinol-binding protein (RBP), but it is not clear how maternal retinol reaches the embryo. We demonstrate here that uptake of radiolabeled retinol from holo-RBP to the visceral yolk sac of cultured rat embryos exhibits receptor-mediated characteristics (Km = 4.1 x 10(-7) M, Vmax = 3.0 x 10(-12) mol/sec per gram yolk sac protein) and also demonstrate the presence in the yolk sac endoderm of a protein previously described as an RBP receptor. Furthermore, transport of radiolabeled retinol from the yolk sac to the embryo proper can be markedly decreased by inhibition of visceral yolk sac RBP synthesis. Thus embryonic accumulation of retinol depends on a relay of retinol from maternal RBP to RBP synthesized in the yolk sac, via interaction with a visceral yolk sac RBP receptor.

The functional basis of tissue-specific retinoic acid signalling in embryos

Retinoic acid (RA) is essential for normal embryonic development. In mammals it is sequestered from the maternal circulation in the form of retinol. In rodents, embryonic uptake relies upon the presence of retinol binding protein (RBP) in the yolk sac, probably involving an RBP receptor. The molecular activity of RA in the nucleus is well established, but less is known about cytoplasmic events including tissue-specific intraembryonic RA synthesis and intracellular transport of both retinol and RA. The cellular binding proteins for retinol and RA may play important roles in these processes.

Retinoid binding proteins in mouse yolk sac and chorio-allantoic placentas.

In the adult, as well as in the embryo, a number of specific extra- and intracellular binding proteins such as the plasma retinol binding protein (RBP), the cellular retinol binding protein type I (CRBP I), and also the cellular receptors for RBP are thought to regulate transport and metabolism of retinol (vitamin A). Since the regulation of materno-fetal transport of vitamin A is not well understood, we examined the localization of these proteins during the development of the mouse chorio-allantoic and yolk sac placentas. The labyrinthine region of the chorio-allantoic placenta, where exchange of substances can occur between the maternal and fetal circulations, did not contain RBP (mRNA or protein) or antigen(s) similar to the bovine RBP-receptor p63, whereas the visceral endoderm of the yolk sac placenta, the second site for materno-fetal transport, did. Furthermore, only the endodermal cells of the visceral yolk sac appeared to strongly accumulate radiolabelled retinoids. The cellular retinol binding protein (CRBP I) was detected both in the trophoblast layer of the placental labyrinth closest to the fetal endothelium (layer III), and in the visceral endoderm of the yolk sac. Together, these findings suggest that the yolk sac placenta mediates retinol transfer to the embryo/fetus throughout the entire gestation. The chorio-allantoic placenta, on the other hand, does not appear to have this capacity, while the presence of CRBP I does suggest a retinol-metabolizing capability.

Prenatal ethanol consumption alters the expression of cellular retinol binding protein and retinoic acid receptor mRNA in fetal rat embryo and brain.

The mechanism by which prenatal ethanol ingestion causes fetal alcohol syndrome (FAS) is unknown. We hypothesize that ethanol disrupts the normal function of retinoids in embryogenesis and differentiation, resulting in FAS. The present work was designed to determine if prenatal ethanol ingestion affects the expression of cellular retinol binding protein (CRBP) and nuclear retinoic acid receptors (RARs). Paired timed pregnant rats were fed a liquid diet, one group treated with 36% of carbohydrate calories replaced with ethanol. Maternal serum retinol concentrations during pregnancy peaked on the 6th day of pregnancy, but no difference was noted between the ethanol and control group. At the 12th and 20th day of gestation, embryos or fetal brain were removed, and RNA was isolated for Northern hybridization. The abundance of CRBP mRNA was significantly elevated by ethanol consumption. In both the 12-day embryo (relative density of control: 1.00 +/- 0.10; vs. ethanol: 1.87 +/- 0.30, p < 0.05) and 20-day fetal brain (relative density of control: 1.00 +/- 0.09; vs. ethanol: 1.46 +/- 0.09, p < 0.01). In the embryo, ethanol ingestion resulted in a decrease in the level of RAR-beta mRNA (control: 1.00 +/- 0.05; vs. ethanol: 0.71 +/- 0.07, p < 0.01), but had no effect on RAR-alpha or RAR-gamma mRNA. In contrast to the embryo, the expression of both the 3.7- and 2.7-kb RAR-alpha transcripts was significantly greater in day 20 fetal brain of ethanol-treated rats (3.7-kb RAR-alpha control: 1.00 +/- 0.11; vs. ethanol: 1.65 +/- 0.06; p < 0.001; 2.7-kb RAR-alpha control: 1.00 +/- 0.14; vs. ethanol: 1.74 +/- 0.27, p < 0.05), whereas RAR-beta and RAR-gamma expression were not altered. These observations suggest that altered vitamin A function is a potential factor in the embryopathy of prenatal ethanol exposure.

The effect of maternal ethanol ingestion on fetal rat heart vitamin A: a model for fetal alcohol syndrome.

Ethanol consumption during pregnancy can cause fetal alcohol syndrome (FAS). Although the exact mechanism is unknown, nutritional alterations caused by ethanol exposure may be an etiologic factor in FAS. The congenital heart defects seen in FAS are similar to those found in vitamin A teratogenesis. Because ethanol ingestion alters vitamin A metabolism, we hypothesized that the cardiac manifestations seen in FAS result from an alteration in vitamin A metabolism or function in the developing fetus. Twenty-day gestation fetal rat hearts from ethanol-exposed and control pregnancies were analyzed for 1) levels of endogenous retinol, retinyl palmitate, and retinoic acid by quantitative HPLC; 2) binding activity levels of both retinol by cellular retinol binding protein and retinoic acid by cellular retinoic acid binding protein using specific competitive binding assays; and 3) relative abundance of cellular retinol binding protein and retinoic acid receptor alpha, beta, and gamma subtype message as expressed in mRNA. Levels of retinol and retinyl palmitate were significantly higher (p < 0.01) and the level of retinoic acid was significantly lower (p < 0.02) in the ethanol-exposed fetal hearts. Binding activity levels of cellular retinol binding protein and cellular retinoic acid binding protein were not different in the two groups. The message for retinoic acid receptor alpha (3.7 kb) was increased (p < 0.01) and the message for retinoic acid receptor beta was decreased (p < 0.05) in the ethanol-exposed hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinol-Binding Protein Mediates Uptake of Retinol to Cultured Human Keratinocytes

Retinol (vitamin A) circulates in the blood bound to retinol-binding protein (RBP). The process by which target cells acquire retinol is not fully elucidated, although a cell surface receptor for RBP has recently been identified. We show here that retinol is at least an order of magnitude more efficient at blocking the terminal differentiation of cultured normal human keratinocytes when administered as a complex with RBP than when administered free in solution. This inhibition of differentiation by RBP can be reversed by monoclonal antibody P142, reactive toward the RBP-binding membrane protein p63. These results demonstrate, at least in this in vitro system, the importance of the RBP receptor in the generation of a cellular response to retinol.

Solubilization and purification of the retinol-binding protein receptor from human placental membranes

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.

Structure-function studies on human retinol-binding protein using site-directed mutagenesis

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

Porcine conceptus and endometrial retinoid-binding proteins

Porcine conceptus secretory proteins were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialysed and concentrated by Amicon ultrafiltration for retinol and retinoic acid (RA) binding studies. Proteins in the 20-kDa range, conceptus-secreted retinol-binding protein (RBP), bound both [3H]retinol and [3H]RA specifically. Cross-competition experiments indicate that [3H]RA was completely displaced with excess cold retinol; however, excess cold RA did not completely displace [3H]retinol, suggesting that conceptus RBP has greater affinity for retinol than RA. Cellular RBP and retinoic acid receptor (RAR)-alpha and RAR-gamma mRNA transcripts (0.7 kb; 3.8 and 2.8 kb; 3.4 kb respectively) were detected in poly (A)+ RNA isolated from Day-15 conceptus, Day-15 pregnant endometrium, late pregnant myometrium and late pregnant fetal tissues of pigs by Northern blot analysis. RAR-alpha and RAR-gamma immunoreactive proteins were detected in extracts of Day-15 conceptus, Day-15 pregnant endometrium and late pregnant fetal tissues by Western blot analysis. Collectively, results indicate that biochemical molecules required for retinoid transport, metabolism and regulatory effects are present in porcine conceptus and endometrial tissues during early pregnancy in swine.

The retinal pigment epithelial membrane receptor for plasma retinol-binding protein. Isolation and cDNA cloning of the 63-kDa protein.

Retinol, a metabolic precursor of retinal and retinoic acid, is transported in plasma by the plasma retinol-binding protein (RBP). The cellular uptake of retinol from RBP is believed to involve a specific membrane receptor for RBP. In retinal pigment epithelium the RBP receptor appears to be an oligomeric protein complex, and we have previously identified a 63-kDa membrane protein as part of this receptor. The 63-kDa protein (p63) has now been isolated, and we have cloned the corresponding cDNA. In a data base search no sequences similar to p63 were identified. Hydropathy analyses of the 533 amino acids deduced from the cDNA sequence did not indicate an N-terminal signal sequence or obvious transmembrane regions. In vitro translation of synthetic mRNA encoding p63, in the presence of heterologous microsomes, verified that p63 does not become cotranslationally membrane-inserted. Transcripts for p63 are abundantly expressed in retinal pigment epithelium with no detectable expression in several other tissues. Southern blotting analysis of bovine and human genomic DNA revealed several hybridizing fragments suggesting a complex organization of the corresponding genes.

Specific uptake of retinol-binding protein by variant F9 cell lines.

Serum retinol-binding protein (RBP) specifically binds to and is internalized by F9 embryonal carcinoma cells. Monolayers of F9 cells were differentiated into a primitive endoderm stage by addition of retinoic acid. Fluorescein-derivatized or radiolabeled RBP associated with F9 cell monolayers at 37 degrees C in a time- and concentration-dependent manner. Competition by simultaneous incubation with excess unlabeled RBP indicated that this association was specific and saturable; the apparent dissociation constant was 200-300 nM using either tracer. At 37 degrees C, over 80% of the cell-associated RBP was internalized, and only a small fraction was bound to the cell surface; fluorescence microscopy indicated that internalized RBP was in small vesicles within the cytoplasm. Internalized RBP was subsequently degraded and released from the cell in an acid-soluble form. Parental F9 cells were heterogeneous in their ability to associate with RBP. Random subcloning identified natural variant F9 cell lines which did, or did not, express this biological activity upon retinoic acid-induced differentiation. The clonal nature of the capacity for RBP uptake suggests that this specific internalization is a heritable trait. Together, these observations provide strong evidence that RBP uptake occurs by a receptor-mediated process in F9 cells. The cycle of RBP internalization and degradation by F9 cells bearing specific RBP receptors may provide a regulable mechanism for the cellular accumulation of serum retinol.

Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells.

Rat liver stellate cells were cocultured with HepG2 human hepatoma cells, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was studied by cryoimmunoelectron microscopy. In stellate cells, human RBP was found on the cell surface and within endosomes. The transfer of human RBP from HepG2 cells to stellate cells was blocked by addition of RBP antibodies to the culture medium. Very little uptake of RBP was observed when fibroblasts were cocultured with HepG2 cells. In a series of experiments, RBP was bound to its putative cell surface receptor at 4 degrees C, and the stellate cells were washed and then incubated at 37 degrees C in order to allow them to internalize a pulse of RBP. About 50% of the RBP was internalized after 6 min of incubation. The RBP-positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membranes. After 2 hr, however, most RBP was localized in intracellular vesicles at a distance from the vesicular membranes, suggesting that RBP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with 125I-RBP at 4 degrees C and cell-associated radioactivity was determined. The calculated dissociation constant for the specific binding was 12.7 +/- 3.2 nM. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cells bound about 30 times more 125I-labeled RBP than did parenchymal cells (based on mass of cell protein). These data suggest that RBP mediates the paracrine transfer of retinol from hepatocytes to perisinusoidal stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis.

Retinol uptake from retinol-binding protein (RBP) by liver parenchymal cells in vitro does not specifically depend on its binding to RBP.

The uptake characteristics of both the retinol and retinol-binding protein (RBP) moieties of the retinol-RBP complex by liver parenchymal cells (PC) in vitro were studied to assess whether retinol uptake is mediated by a cell-surface receptor for RBP. At 37 degrees C as well as 4 degrees C, [3H]retinol uptake from [3H]retinol-RBP showed a time-dependent increase, and was not saturable at concentrations exceeding the physiological concentration by more than a factor of 2 (3 microM). Uptake of [3H]retinol was not inhibited by a 10-fold molar excess of unlabeled retinol-RBP. Cell association of 125I-RBP at 37 and 4 degrees C was low and showed no time dependence. In addition, the association of 125I-RBP was not saturable at concentrations up to 3 microM. These data do not support the existence of a cell-surface receptor for RBP on rat liver PC. The uptake of [3H]retinol from RBP was also compared to the uptake of retinol from cellular retinol-binding protein (CRBP) and lactoglobulin. Uptake characteristics of [3H]retinol from CRBP and lactoglobulin were similar to that of [3H]retinol from RBP. Furthermore, a similar percentage of the [3H]retinol taken up by PC was metabolized into retinyl esters, irrespective of its carrier. These data suggest that the uptake of retinol and its subsequent metabolic processing do not depend on binding to RBP. The low level of cell association of 125I-binding proteins was not due to uptake, degradation, and secretion of ligand by PC. This suggests that retinol is dissociated from its binding protein before uptake by PC.

A Comparison of the Uptake, Metabolism and Biologic Effects of Retinol Delivered to Human Keratinocytes Either Free or Bound to Serum Retinol-Binding Protein

In response to body demands, retinol (ROL) is secreted from the liver into the circulation bound to serum retinol-binding protein (RBP). The mechanism by which ROL is transferred from RBP to target cells remains controversial. To study ROL delivery, we have used a model system of cultured human foreskin keratinocytes (HKc) to compare the uptake, metabolism and biologic effects of ROL added either directly to the medium or bound to RBP. [3H]ROL added directly to the medium was rapidly taken up by HKc, and maximal accumulation of [3H]ROL occurred by 3 h. In contrast, [3H]ROL delivered bound to RBP was taken up very slowly but at a linear rate for at least 72 h. Several experimental approaches indicated that ROL uptake from RBP was not mediated by a cell surface receptor for RBP. The rate and extent of [3H]ROL metabolism to retinyl esters was the same whether the ROL was added directly to the medium or bound to RBP. In addition, several biologic responses by HKc to ROL showed the same dose response curves whether the ROL was added directly to the medium or bound to RBP. Overall, the results support a model of ROL uptake from RBP in which the vitamin is first slowly released from RBP into the aqueous phase and then becomes cell associated. In this manner, the cells are provided with a slow but constant supply of ROL and are protected from the rapid and potentially toxic accumulation of ROL that occurs in the absence of RBP.

Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium

A specific membrane receptor for plasma retinol-binding protein (RBP) is expressed in the retinal pigment epithelium (RPE). When chemically cross-linking RBP to RPE membranes, an 86-kDa RBP.RBP receptor complex is formed, and a 63-kDa protein was identified as the RBP-binding membrane protein (Båvik, C.-O., Eriksson, U., Allen, R., and Peterson, P. (1991) J. Biol. Chem. 266, 14978-14985). To explore in more detail the characteristics of this membrane receptor, we have generated a monoclonal antibody, A52, to the 63-kDa protein (p63). A52 binds the 86-kDa RBP.RBP receptor complex and p63. Several lines of evidence suggest that p63 is not a regular integral membrane protein, and it occurs in different forms. One form is firmly attached to membranes, is part of a high molecular weight complex, and is able to bind RBP. The other form of p63 can be removed from membranes by treatment with an alkaline buffer and is unable to bind RBP. Both forms of p63 contain extensive hydrophobic domains and are found in the detergent phase upon extraction with Triton X-114. The expression of p63 is restricted to RPE, and immunohistochemical localization of tissue sections from bovine retina showed highest expression in the basolateral portion of RPE cells. Immunofluorescence localization, using isolated RPE cells, showed that p63 is exposed on the cell surface of newly isolated RPE cells.

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.

Comparison of the Rate of Uptake and Biologic Effects of Retinol Added to Human Keratinocytes Either Directly to the Culture Medium or Bound to Serum Retinol-Binding Protein

Retinol circulates in the plasma bound to retinol-binding protein (RBP), but the mechanism by which retinol is transferred from RBP to target cells is not known. To study retinol delivery, human keratinocytes (HKc) were incubated with [3H]retinol added directly to the culture medium or bound to RBP and the uptake of [3H]retinol was determined at various times. During the first hour of incubation, the rate of [3H]retinol accumulation by HKc was about 40 times greater when the vitamin was added directly to the media rather than bound to RBP. Although maximal uptake of [3H]retinol added directly to the culture medium occurred at 3 h, the uptake of [3H]retinol from RBP was linear with time for at least 72 h. By 57 h, cell-associated [3H]retinol was the same whether it was added directly to the culture medium or bound to RBP. Excess unlabeled retinol or pretreatment of HKc with retinol had no effect on the uptake of [3H]retinol added directly to the culture medium or bound to RBP. Apo- but not holo-RBP was capable of competing with HKc for the uptake of [3H]retinol from RBP. No specific or saturable binding of 125I-labeled RBP to HKc cultured in the absence or the presence of retinol was found. The dose response of retinol inhibition of cholesterol sulfate synthesis and phorbol ester-induced ornithine decarboxylase activity or retinol modulation of keratin expression was the same whether the retinol was delivered to HKc bound to RBP or added directly to the medium. Our data support a mechanism for retinol delivery from RBP to HKc that does not involve cell-surface RBP receptors but instead suggest that the vitamin is first slowly released from RBP and then becomes cell-associated from the aqueous phase. This mechanism is consistent with the finding that HKc respond identically to retinol whether or not it is delivered to them bound to RBP.