Human Retinol-binding Protein Research Articles

The role of extrahepatic retinol binding protein in the mobilization of retinoid stores

Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP(-/-)) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP(-/-)). By comparing hRBP(-/-), RBP(-/-), and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical. Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Muscle Expression of Human Retinol-binding Protein (RBP): SUPPRESSION OF THE VISUAL DEFECT OF RBP KNOCKOUT MICE

Mice lacking retinol-binding protein (RBP) have low circulating retinol levels. They have severe visual defects due to a low content of retinol or retinyl esters in the eye. A transgenic mouse strain that expresses human RBP under the control of the muscle creatine kinase promoter in the null background was generated. The exogenous protein bound retinol and transthyretin in the circulation and effectively delivered retinol to the eye. Thus, RBP expressed from an ectopic source suppresses the visual phenotype, and retinoids accumulate in the eye. No human RBP was found in the retinal pigment epithelium of the transgenic mice, indicating that retinol uptake by the eye does not entail endocytosis of the carrier RBP.

Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci

Genetic alterations associated with human hepatocellular carcinoma (HCC) have been reported previously, but are not sufficient to specify differences of HCCs from precancerous diseases of the liver, such as hepatitis, hepatic fibrosis, and cirrhosis. In the present study, we performed differential gene display analysis (DGDA) to clarify the specific genetic alterations associated with gene expression changes in the course of development of HCC from chronic viral hepatitis. Four pairs of surgically resected HCCs and hepatitis tissues were investigated. We found 1,028 expression sequence tags (ESTs) that were decreased or increased in HCC tissues compared with hepatitis tissues in the same patient. Nucleotide sequencing showed that they included 55 EST clones in the GenBank database, which were considered candidates for specific messenger RNA (mRNA) expression alterations in HCCs. After excluding 9 ESTs that code mitochondrial DNA, we performed quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) for the 46 remaining EST clones. We found 8 mRNAs underexpressed in primary HCC tissues in 20 patients in higher percentages than found in previous studies, including 18 cases (90%) for aldolase B (ALDOB), 15 cases (75%) for carbamyl phosphate synthetase 1 (CPS1), albumin (ALB), plasminogen (PLG), and EST 51549, 13 cases (65%) for cytochrome P450 subfamily 2E1 (CYP2E1), 12 cases (60%) for human retinol-binding protein 4 (RBP4), and 11 cases (55%) for human organic anion transporter C (OATP-C) gene. In conclusion, underexpression of key gene products may be important in the development and/or progression of HCC. (H EPATOLOGY 2002;36:433-438.)

Developmental expression, tissue distribution and hormonal regulation of fish (Sparus aurata) serum retinol-binding protein

Retinol-binding protein (RBP) is the specific carrier of retinol in vertebrates and forms a 1:1 complex with transthyretin (TTR). A cDNA encoding serum RBP was cloned from liver and 7-day larvae of the marine fish Sparus aurata. The mature protein is 176 amino acids long and shows sequence identity of 77–78%, 56%, 63% and 62% with rainbow trout, Xenopus, chicken and human RBP, respectively. Northern blot analysis of hepatic RBP revealed two transcripts: a major one of approximately 1.4–1.5 kb and a minor of approximately 0.7 kb. Distribution of RBP mRNA in various tissues was studied by RT-PCR and showed high expression in liver and skin, and low expression in brain, kidney and gill filament (20–35% of the level in liver). RBP expression in intestine, pyloric caeca, muscle and pituitary was estimated to be approximately 7–14% of the level in liver. The ontogeny of RBP expression in S. aurata was examined in unfertilized eggs, embryos and larvae by using RT-PCR followed by hybridization with a specific probe. RBP transcript was found in all larval stages studied. Very low levels of RBP mRNA were detected in unfertilized eggs and in embryos 8 h after fertilization with a gradual increase at 12 h and 15–16 h post-fertilization. A single injection of estradiol-17β to S. aurata immature, bisexual fish or to adult males reduced steady-state levels of hepatic RBP by 37 and 25%, respectively. The same treatment induced vitellogenin expression. The present data suggest that in fish, liver is the main site of RBP synthesis, but that RBP may have an important function in fish skin. RBP is expressed early in embryonic development and in fish its expression can be down regulated by estrogen.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein.

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified iota-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282-3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (K(d) approximately 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-A x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

IRBP Enhances Removal of 11- cis -retinaldehyde from Isolated RPE Membranes

Interphotoreceptor retinoid-binding protein (IRBP) greatly enhances the conversion of all-trans -retinol to 11- cis -retinal by the retinal pigment epithelium (RPE) and facilitates 11- cis -retinal release from the RPE. However, the mechanisms by which IRBP exerts these effects are not clear. Using a model system of purified bovine IRBP and isolated bovine RPE membranes, we investigated the possibility that IRBP may favor the delivery of all-trans -retinol to, or the release of 11- cis -retinal from, RPE membranes. As the interphotoreceptor space contains serum retinol-binding protein (RBP) and serum albumin in addition to IRBP, we similarly examined the exchange of retinoids between RPE membranes and human RBP or bovine serum albumin (BSA). Isolated RPE membranes were loaded with radioactive 11-cis -retinal and incubated with solutions of IRBP, RBP, BSA or with buffer alone. Membranes (pellet) and retinoid-binding protein or buffer (supernatant) were separated by centrifugation and analysed for radioactive 11- cis -retinal. Membranes incubated with buffer alone released only 4–5% of their 11- cis -retinal, while 25 μm IRBP removed 18–35%. More retinal was released as the membrane concentration was reduced. In contrast, RBP and BSA removed little retinal, even though both proteins are capable of binding this retinoid. Similar results were obtained with bovine liver membranes, consistent with the idea that the effects of IRBP do not depend on an RPE surface receptor for IRBP. IRBP was also markedly superior to RBP and BSA in removing all- trans -retinol from RPE membranes. In addition, IRBP efficiently delivered bound all- trans -retinol to membranes; however, in contrast to their differential removal of retinoids, all three binding proteins delivered comparable amounts of retinol to membranes. (This result supports the practice of using BSA as a retinoid carrier in in vitro experimental systems). We conclude that, whereas IRBP shares with other retinoid-binding proteins the ability to deliver retinol to membranes, IRBP is unique in its capacity to remove 11- cis -retinal from membranes. This may be the feature of IRBP that drives the vitamin A cycle to efficiently produce 11- cis -retinal.

Mouse retinol binding protein gene: cloning, expression and regulation by retinoic acid.

A full-length cDNA clone encoding the retinol binding protein (RBP) was isolated from a mouse liver cDNA library by hybridization screening. The nucleotide sequence of murine RBP is 85 and 95% homologous to that of human and rat RBP, respectively, with a deduced amino acid sequence > or = 83% homologous to both species. Analysis of the tissue expression pattern of RBP mRNA in the female mouse indicated relatively abundant expression in the liver, with lesser amounts in extrahepatic tissues including adipose, kidney, spleen and uterus, suggesting that these tissues may have a significant role in retinol homeostasis. Mouse liver cell RBP regulation by retinoids was also investigated. Both all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9c-RA) induced RBP mRNA expression in a dose- and time-dependent manner. Maximal levels (up to 4-fold above controls) were observed at > or = 48 h following treatment of both mouse hepatoma cells in vitro and in vivo in mice receiving a single, oral dose of either retinoid. Interestingly, 9c-RA was more potent at RBP induction in both in vivo and in vitro systems. Given the extent and temporal pattern of RBP induction, we suggest that the RA-mediated increase in liver RBP is part of a cellular protection mechanism. Increased levels of RBP would facilitate sequestration and possibly cellular export of RA in cells receiving prolonged exposure to high levels of RA, thus minimizing toxicity.

The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP.

Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.

Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP Fab.

A macromolecular complex of human transthyretin, human retinol-binding protein and an anti-retinol-binding-protein Fab was crystallized by vapour diffusion in sitting drops. Diffraction from these crystals at cryogenic temperatures was consistent with the space group C222, with cell parameters a = 159.34, b = 222.40 and c = 121.27 A. Crystals diffracted to a resolution limit of 3.36 A using synchrotron radiation. Based on a 2:2:1 stoichiometry for the Fab-retinol-binding-protein-transthyretin complex and the presence of one such complex per asymmetric unit, a reasonable Vm coefficient of 2.74 A3 Da-1 could be estimated.

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Human retinol-binding protein (RBP) is a monomeric 21-kDa protein that is currently the subject of numerous studies owing to its role in the cellular uptake and utilization of retinol. When the RBP gene is overexpressed inEscherichia coli,inclusion bodies of aggregated RBP are found in the cells. These inclusion bodies are solubilized in 5.0 M GdmCl containing 10 mM DTT. Refolding of RBP is carried out in the presence of vitamin A by diluting denatured and reduced RBP into a redox refolding buffer consisting of 3 mM cysteine/0.3 mM cystine at 4°C. Ion exchange chromatography (HPLC) is utilized to purify refolded RBP to homogeneity as demonstrated by SDS–PAGE and electrospray MS. The native structure of refolded RBP was established by its ability to bind to vitamin A and the plasma protein transthyretin. The reconstitution of RBP outlined within affords a 50–60% overall yield, i.e., 73 mg of pure RBP/L ofE. coliculture.

Retinol-induced secretion of human retinol-binding protein in yeast

Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol. Thus, the molecular mechanism involved in retinol-dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol-regulated secretion of RBP. RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays.

Retinol free and retinol complexed β-lactoglobulin binding sites in bovine germ cells

A high affinity specific binding site for bovine β-lactoglobulin (BLG) was identified in bovine germ cell plasma membrane enriched fractions. Binding was found to be reversible and pH-dependent with maximum binding occurring at pH 5. The on-rate and off-rate constants were 2.26±0.8×10 5 M −1 min −1 ( n=3) and 0.016±0.004 min −1 ( n=3), respectively. Scatchard analysis showed a single class of binding sites, with 12.38±4.62×10 12 sites per mg of membrane protein ( n=3) and a dissociation constant ( K D) estimated at 26.43±2.68 nM. There was inhibition of iodinated-BLG (variant A) ( 125I-BLGA) binding to germ cell plasma membrane enriched fractions in the presence of unlabelled BLG variant A, BLG variant B, retinol complexed BLGA and human retinol-binding protein. Inhibition was observed neither with BSA nor with lactoferrin. 125I-BLGA incubated with a Triton X-100 solubilized plasma membrane fraction formed a high molecular mass complex in Superose 12B gel filtration. This receptor complex disappeared in the presence of unlabelled BLGA and in the presence of 10 mM EDTA. The results suggest that germ cell plasma membrane may contain a receptor which is capable of binding either retinol free or retinol complexed BLGA.

Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin

The retinol carrier retinol-binding protein (RBP) forms a complex with the thyroid hormone binding protein transthyretin in the plasma of a number of vertebrate species. The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with transthyretin have been investigated by means of fluorescence anisotropy studies. The presence of two independent and equivalent RBP binding sites per transthyretin molecule has been established for proteins purified from species distant in evolution. Although the natural ligand retinol participates in the interaction between retinol-RBP and transthyretin, its binding to RBP is not a prerequisite for protein—protein interaction. The dissociation constants of human transthyretin binding liganded and unliganded forms of human RBP were determined to be: all- trans retinol-RBP, K d ∼ 0.2 μM; apoRBP, K d − 1.2 μM; all- trans retinoic acid-RBP, K d − 0.8 μM; all- trans retinyl methyl ether-RBP, K d ∼ 6 μM. The complex of RBP with the synthetic retinoid fenretinide, which bears the bulky hydroxyphenyl end group, exhibits negligible affinity for transthyretin. The replacement of RBP-bound retinol with synthetic retinoids affects RBP-transthyretin recognition to an extent that appears to be well correlated with the nature and steric hindrance of the groups substituting the retinol hydroxyl group, consistent with their location at the interface between the contact areas of RBP and transthyretin.

Purification and properties of porcine allantoic fluid retinol-binding protein

Retinol-binding protein (RBP) was purified from Day 60 porcine allantoic fluid by a combination of diethylaminoethyl cellulose, G-100 Sephadex, G-50 Sephadex, Phenyl-Sepharose, and Reactive Green 19-dye-agarose chromatography. The yield was 1 to 2 mg of RBP, which generated a single M r 20,000 band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and up to four isoelectric variants (isoelectric variants 1 to 4) after two-dimensional PAGE (2D-PAGE). The protein cross-reacted with antiserum raised against human RBP. When incubated with [ 3H]retinol and subjected to G-100 Sephadex chromatography, [ 3H]retinol coeluted with the protein. These results indicate that the purified protein is an RBP. When purified RBP was subjected to native 2D-PAGE, six forms of RBP were observed. Three native forms were fluorescent, and three were not fluorescent, suggesting that these forms were RBP with and without retinol, respectively. Denaturing 2D-PAGE analysis of each native form of RBP suggested that two of the nonfluorescent and two of the fluorescent native forms of RBP corresponded to isoelectric variant 1 on denaturing 2D-PAGE, whereas the other fluorescent and nonfluorescent forms corresponded to isoelectric variant 2. The incubation of RBP with 50 μM retinol enhanced the amount of both isoelectric variants present as fluorescent RBP, but uptake by isoelectric variant 1 was greater than that by isoelectric variant 2. These data indicate that RBP can be purified from porcine allantoic fluid and suggest that the isoelectric variants may differ in their affinity for retinol.

Purification and characterization of a uterine retinol-binding protein in the bitch.

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

Lutein associated with a transthyretin indicates carotenoid derivation and novel multiplicity of transthyretin ligands

Transthyretins isolated from different species bind hydrophobic compounds and are often obtained in a yellow form. Such a transthyretin from chicken serum was purified by chromatography using Sepharose-coupled human retinol-binding protein. The yellow chromophore was extracted with methanol and purified by reverse phase HPLC followed by normal-phase chromatography on a nitrile column. Ultraviolet-visible absorbance and mass spectrometry identified the yellow compound as lutein, i.e. xanthophyll, ( all-trans)-β,ϵ-carotene-3,3′-diol, estimated to constitute 10–30% of associated colourless compounds. These components are different from the yellow component isolated from human transthyretin and establish that carotenoid-derived pigments can be associated with transthyretins.

Epitope Mapping of a Monoclonal Antibody That Blocks the Binding of Retinol-Binding Protein to Its Receptor

To define the receptor binding site of retinol-binding protein (RBP) we have generated monoclonal antibodies (mAbs) to human RBP and examined their ability to interfere with the receptor binding. MAbs to two conserved regions efficiently blocked the binding. No major conformational changes in the protein occurred upon mAb binding, since the mAbs could co-immunoprecipitate the RBP-transthyretin (TTR) complex. One blocking mAb showed reactivity to a synthetic peptide corresponding to one entrance loop of the retinol binding pocket (amino acid residues 60-70). Thus, our results show that at least one of the entrance loops of the barrel of RBP is located in or close to the receptor binding site. It can also be concluded that the receptor and TTR binding sites involve different regions of RBP.

The Ile-84–>Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinol-binding protein.

In plasma the thyroid hormone-binding protein transthyretin (TTR) forms a tight complex with the specific retinol carrier retinol-binding protein (RBP). The Ile-84-->Ser mutation and several other point mutations in TTR are associated with familial amyloidotic polyneuropathy, which is characterized by extracellular depositions of amyloid fibrils mainly consisting of mutated TTRs. The interactions with human RBP of recombinant human normal and Ser-84 TTRs were investigated by monitoring the fluorescence anisotropy of RBP-bound retinol. A nearly negligible affinity of the recombinant Ser-84 TTR for RBP was found. This result indicates the participation of a region on the outer surface of TTR that comprises Ile-84 in the recognition of RBP. In preliminary studies the Ser-84 TTR was the only one among several amyloidogenic variant TTRs to display negligible interaction with RBP. Therefore, in general a substantially altered binding of TTR to RBP is not associated with familial amyloidotic polyneuropathy. Instead, the altered binding of Ser-84 TTR to RBP appears to be responsible for an abnormal plasma transport of RBP. The recombinant normal TTR exhibits binding properties, in its interaction with human RBP, approximately similar to those of TTR purified from human plasma. Two independent and equivalent RBP binding sites on recombinant normal TTR are characterized by a dissociation constant of about 0.4 microM.

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.