FASEB J. Uncover Your Biological Pathway
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-8939com.
 (The FASEB Journal. 2008;22:1043-1054.)
© 2008 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.07-8939comv1
22/4/1043    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Redondo, C.
Right arrow Articles by Findlay, J. B. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Redondo, C.
Right arrow Articles by Findlay, J. B. C.
(The FASEB Journal. 2008;22:1043-1054.)
© 2008 FASEB

Identification of the retinol-binding protein (RBP) interaction site and functional state of RBPs for the membrane receptor

Clara Redondo*, Maria Vouropoulou*, Jonathan Evans* and John B. C. Findlay*,1

* Institute of Membrane and Systems Biology, Faculty of Biological Sciences, LIGHT Laboratories, University of Leeds, Leeds, UK

1Correspondence: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, LIGHT Laboratories, Clarendon Way, University of Leeds, Leeds LS2 9JT, UK. E-mail: j.b.c.findlay{at}bmb.leeds.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This laboratory has advanced a model whereby retinol is transported around the body bound to retinol-binding protein (RBP), is transferred across the membrane of cells by a specific receptor/transporter, and is picked up from the membrane by an intracellular homolog, cellular retinol-binding protein (CRBP). This process involves a number of protein-protein interactions, and we hypothesized that conformational changes were an integral part of the retinol transfer mechanism. Previously we identified the potential interaction site on RBP for its membrane receptor. Here we confirm by the analysis of chimera containing a grafted CD loop from RBP that this is indeed the receptor interaction site and go on to demonstrate that the conformational changes that occur to this region on the apo to holo transition in RBP also take place in a chimera binding a quite different ligand, thus establishing the concept. We have also gone on to support the hypothesis that CRBP may also bind to a receptor in the membrane. Previous evidence has indicated that one such receptor might be lecithin:retinol acyltransferase, an enzyme that catalyzes retinol esterification. Here we provide the first evidence that the plasma membrane receptor for RBP could be the same as that for CRBP. This observation offers support for the intracellular phase of the uptake process for retinol, providing an efficient and highly unique mechanism in eukaryotic biology.—Redondo, C., Vouropoulou, M., Evans, J., Findlay, J. B. C. Identification of the retinol-binding protein (RBP) interaction site and functional state of RBPs for the membrane receptor.


Key Words: apo • holo • surface plasmon resonance • mouse urinary protein • lipocalin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RETINOL, THE ALCOHOL FORM of vitamin A, is an essential nutrient for growth, development, reproduction, and vision and is transported in plasma to target-cells bound to a specific carrier protein, retinol-binding protein (RBP). RBP is synthesized primarily in the liver, where it requires the binding of all-trans-retinol to trigger its secretion as a complex with transthyretin (TTR). Other sites of synthesis are the kidney, peritubular and Sertoli cells of the testis, the retinal pigment epithelium, adipose tissue, and the choroid plexus of the brain (1 , 2) .

In mammals, RBP circulates as a monomer (21 kDa) in equilibrium with a coupled form involving a tetramer (56 kDa) of TTR. The TTR:RBP complex (~77 kDa) prevents RBP from being lost through glomerular filtration and encapsulates retinol, restricting it from freely partitioning into the plasma membrane.

A specific receptor for RBP in the plasma membrane of many eukaryotic tissues has been reported in placenta (3) , Sertoli cells (4) , stellate cells (5 , 6) , peritubular pigment epithelial and embryonic carcinoma cells (7) , choroid plexus (8) , and a variety of tissue culture cells (9 , 10 ; as reviewed in refs. 11 , 12 ). The free rather than the complexed form of RBP interacts with the receptor.

Sundaram et al. (13) first advanced the theory, based on transport studies, that this cell surface receptor mediated the transfer of retinol from RBP in the extracellular compartment to one of several structurally related cellular retinol-binding proteins (CRBPs) found within the aqueous environment of the cytosol. Since then more recent work with RBP knockout mice has supported this natural receptor-mediated retinol uptake system (14 15 16 17) . Very recently, Kawaguchi et al. (18) reported the identification in bovine retinal pigment epithelial cells of an integral membrane protein as a specific receptor for RBP. Mass spectrometry analysis of the RBP-receptor complex revealed that the RBP membrane receptor was a "stimulated by retinoic acid gene 6 (STRA6) homolog (mouse)," without homology to any protein of known function. They confirmed that it mediates the cellular uptake of retinol from RBP. Particularly strong expression of STRA6 occurred in cells that compose human blood-organ barriers (e.g., the brain, eye, testis, kidney, spleen, and female reproductive tract).

RBP was the first member of the lipocalin superfamily for which an X-ray structure was described (19) . The basic framework is an eight-stranded antiparallel β-barrel (β-strands named A–H) attached to which is a carboxyl-terminal {alpha}-helix. The amino terminus of the protein, which includes one of the three regions of highly conserved amino acids found in lipocalins, seals one end of the barrel, whereas the opposite end is the entrance to the ligand-binding cavity. The loops connecting the β-strands vary in length, with that joining the A and B strands being the longest and folding over the open end of the barrel.

All-trans-retinol binds to RBP with the isoprene tail fully extended in the β-barrel and the trimethyl cyclohexylene ring innermost in a tight steric fit in the hydrophobic binding pocket. Aromatic amino acids are abundant at the ring end of the ligand, whereas its hydroxyl is positioned at the protein surface, in the region of the entrance loops surrounding the opening of the binding cavity, where it participates in polar interactions. The high degree of complementarity in the shape of the binding site and the structure of retinol is consistent with the fact that RBP is less tolerant of changes to the ring end of the ligand structure where structural differences would be difficult to accommodate without the rearrangement of amino acids in the protein core.

Structure-function studies (20) , using mutants of RBP, showed that loop AB was involved in the RBP-TTR interaction, whereas loops CD and EF, particularly CD, were heavily involved in RBP binding to the placental receptor. Indeed, the CD loop that guards the entrance to the binding pocket, without any real contacts with the ligand, was found to be the major determinant for receptor recognition and interaction (21) . The hypothesis advanced in Sundaram et al. (13) and subsequent reviews (13 , 22 , 23) involves a high-affinity retinol binding form of RBP interacting with its receptor and releasing retinol to the transport mechanism. In so doing, RBP assumes a lower-affinity form, which can readily be replaced on the receptor. RBP isolated from serum has previously been shown to consist of two forms, one able to bind to the placental receptor with high affinity and the other with low affinity (3) . The higher-affinity binding form is thought to be converted into the lower-affinity state on detaching from TTR, binding to the receptor, and releasing retinol. This implies some type of conformational communication between the ligand-binding site and the CD loop that interacts with the receptor. To investigate this hypothesis and to confirm the identification of the receptor binding region, we transferred this loop to another lipocalin [mouse urinary protein (MUP)] and examined its receptor binding properties using a ligand quite different from retinol.

MUP2 is one of a series of variants of the major urinary protein, which is an abundant pheromone-binding protein found in male mouse urine, where subtle recognition of a series of related compounds is thought to be involved in its biological function. The major site of MUP synthesis is the liver from which it is secreted into serum where it circulates at relatively low concentrations before being filtered by the kidney and excreted into urine. Expression of MUP mRNA is under different development and hormonal control in different tissues. Unlike RBP, MUP does not bind retinoids but a spectrum of small odorant molecules and pheromones (24) , and its ligand-binding site is deeper and wider than the one for RBP. Among a number of odorant molecules MUP was reported to bind with the highest affinity to 2-isobutyl-3-methoxypyrazine (IBMP), methyldihydrojasmonate, methylfenchol, and a number of unsaturated cyclic aliphatic compounds (25) . Furthermore, MUP differs from RBP in the length of the β-strands as well as the conformations and lengths of the loop regions.

Here, we explore whether the receptor-binding properties of RBP could be transferred to a quite different member of the same family, MUP, with the grafting of loop CD and using a ligand other than retinol. We further compared the ligand-binding behavior of the apo and holo forms of the chimeric protein [mouse urinary protein with grafted CD loop from RBP (MUPcd)] with RBP to examine whether there are conformationally dependent interactions and whether any functional differences could be related to structural differences. Based on crystallographic data for the apo and holo forms of MUPcd, we propose a conformation-driven model for the specific interaction of RBP with its membrane receptor initiated by the presence of the ligand facilitated by appropriate positioning of the CD loop.

The second phase of the uptake process involves transfer of retinol to CRBP. The mechanism of this transfer is unknown. To increase our understanding, studies were undertaken to determine whether CRBP also exhibited specific interactions with the membrane and, if so, to develop more fully the hypothesis of whether the receptors for RBP and CRBP were the same protein. This article describes progress on both these issues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Grafting of RBP CD loop into MUP
The MUP chimera (MUPcd) was generated in the pQE30 vector (Qiagen, Valencia, CA, USA) using a three-arm ligation into the BamHI and HindIII restriction sites. Two DNA fragments corresponding to the N terminus of MUP with the first half of the CD loop from RBP or to the C terminus of MUP fused to the second half of the CD loop (at the end or at the beginning, respectively) were generated by polymerase chain reaction (PCR) using the Expand High Fidelity PCR System (Roche Diagnostics, Indianapolis, IN, USA). Ligation of both PCR fragments (BamHI and HindIII digested) was concurrent with their ligation into pQE30 (BamHI and HindIII digested), thus ensuring correct orientation. The primer pairs used for the generation of each MUP chimera fragment were N-MUPcd (sense), 5'-CGGATAACAATTTCACACAG-3'; N-MUPcd (antisense), 5'-AAGACGGACTCGGCCATGGAATTTAAGAACTAAGG-3'; C-MUPcd (sense), 5'-GTTCTGAGGTCATTACTGG-3'; and C-MUPcd (antisense), 5'-TTGAATAACTGGGACTGCTCCGAATTATCTATGG-3'. Ligation mixtures were used to transform XL1 Blue Escherichia coli supercompetent cells (Stratagene, La Jolla, CA, USA), and plasmid DNA isolated from the transformants was sequenced to identify the correct chimera sequences. Chimera design was based on secondary structure information from the CATH hierarchical domain classification of protein structures in the Brookhaven Protein Database (PDB) (1mup and 1rbp). The primary protein sequences were obtained from UniProtKB/Swiss-Prot, and throughout this work residue 1 for both proteins (marked with an asterisk) is the first residue after the signal sequence (underlined). MUP residues 58–63 were replaced by grafting RBP residues 59–68 (highlighted in bold) as follows: for MUP, [MKMLLLLCLGLTLVCVHAE*EASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLEKSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE] [P11589 (MUP2_MOUSE)]; and for RBP, [MKWVWALLLLAALGSGRAE*RDCRVSSFRVK ENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNLL] [P02753 (RETBP_HUMAN)].

Expression and purification of recombinant lipocalins
cDNAs of RBP, Mup wild-type (MUPwt), and MUPcd were cloned in pQE30 vector (Qiagen), whereas CRBP [P09455 (RET1_HUMAN)] was cloned into pQE30-Xa (Qiagen). All N terminus hexa-His tagged constructs were expressed in BL21-Gold E. coli cells (Stratagene) and purified using Ni-NTA chromatography (Qiagen). Both recombinant MUPs and CRBP were soluble, whereas RBP was expressed as insoluble protein and had to be refolded and purified from inclusion bodies. Competent E. coli BL21 cells were transformed with expression vector constructs containing RBP, CRBP, MUPwt, or MUPcd. Large-scale expression of the clones was performed from overnight starter cultures, which were grown for a further 3–5 h (A600 of 0.6–0.8). Cultures were induced by the addition of isopropyl-β-D-thiogalactoside (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 1 mM and incubated for 4 h at 30°C, and cells were harvested. To release soluble proteins (CRBP, MUPwt, and MUPcd) from the periplasmic space, cell pellets were lysed with lysozyme (Sigma-Aldrich) (0.4 mg/ml in PBS, pH 7.4) and sonicated, and the final clarified supernatant was incubated with Ni-NTA slurry for purification of tagged proteins. To release recombinantly expressed RBP from inclusion bodies after cell lysis, inclusion bodies were resuspended in a guanidinium chloride (6 M) (Sigma-Aldrich) buffer, and after a 1-h incubation at 4°C, a slow dialysis method against PBS, pH 7.4, was used for refolding. The mixture was clarified by centrifugation, and the supernatant was incubated with Ni-NTA slurry (previously equilibrated in the same buffer) for purification of recombinant RBP. All bound proteins were eluted with 0.5 M imidazole (Sigma-Aldrich) in PBS and pooled fractions were dialyzed against PBS, pH 7.4. For CRBP, dialysis was performed against Xa reaction buffer, and further Xa digestion and purification of cut protein were performed according to manufacturer’s instructions (Qiagen). The concentrations of the purified recombinant proteins were determined by measuring the absorbance at 280 nm using the molar extinction coefficients: 46,760 M–1 cm–1 for RBP, 24,070 M–1 cm–1 for CRBP, and 16,500 M–1 cm–1 for MUP and the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL, USA).

Membrane preparations
A human embryonic kidney cell line (HEK 293) (Clontech Laboratories, Inc., Palo Alto, CA, USA), known to express the RBP receptor (S. Wang and J. B. C. Findlay, unpublished results, 2000), was propagated as a monolayer and grown at 37°C with 5% CO2, in Dulbecco’s modified Eagle’s medium with GlutaMAX-1 supplemented with 2% penicillin/streptomycin and 10% FBS (Sigma-Aldrich). Membranes were prepared by harvesting a density of 107 cells grown in 175-cm2 flasks to 80% confluence. Harvested cells were lysed in the presence of protease inhibitors (Complete Mini, EDTA-free; Roche Diagnostics), and crude membranes were collected by ultracentrifugation at 50,000 rpm for 1 h at 4°C before resuspension in 2-(N-morpholino)ethane sulfonic acid (MES) buffer (25 mM MES/0.15 NaCl, pH 6.5) (Melford Laboratories Ltd., Suffolk, UK) containing 1% Triton-X 100 (v/v) (Sigma-Aldrich). The final supernatant collected as the solubilized membrane preparation was divided into aliquots and stored at –70°C. When Biacore analysis was performed, the aliquots were diluted 1:10 with eluent buffer before binding analysis (final concentration of Triton X-100 was 0.1%). Human red blood cell membranes were prepared according to the method of Dodge et al. (26) . Packed red cells were washed four times with 10 vol of 150 mM NaCl and 10 mM phosphate, pH 7.2, at 4°C, and the supernatant and white cell "buffy coat" were removed by aspiration each time. The cells were lysed in 10 vol of ice-cold 5 mM sodium phosphate, pH 8.0 [containing 0.1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich)], and centrifuged at 15,000 rpm for 20 min. The supernatant was carefully removed, and the process was repeated until the membranes were light pink (three times). These membranes were suspended in ice-cold extraction buffer and treated as above before binding assays.

Placental microvilli membranes were prepared as described in ref. 3 .

Binding assays
Ligand binding
Binding of IBMP to MUP and MUPcd was assessed by isothermal titration calorimetry (ITC) using a MicroCal VP-ITC unit operating at 308 K. Before use, proteins were precipitated with ethanol to remove any endogenous ligands and then redissolved, dialyzed against PBS (pH 7.4), and degassed in a vacuum. The ligand was dissolved in degassed PBS, and all concentrations were measured by UV absorption (MUP-I {epsilon}280=16,500 M–1 cm–1; IBMP {epsilon}220=4980 M–1 cm–1) immediately before titrations were started. The protein concentration was 145 µM, and the IBMP concentration was 1.5 mM. Titrations were performed in duplicate and typically comprised 25 injections (one 2-µl injection followed by 24 5-µl injections) at 4-min intervals. The initial data point was routinely deleted to allow for diffusion of ligand/receptor across the needle tip during the equilibration period. Heats of dilution for the ligand were measured independently and subtracted from the integrated data before curve-fitting in Origin 5.0 with the standard One Site model supplied by MicroCal, which is based on the Wiseman isotherm (27 , 28) :

Formula
where, dQ/d[X]t is the stepwise change in heat of the system normalized with respect to the change in the total concentration of the ligand ([X]t), {Delta}H° is the standard enthalpy for reaction, V0 is the effective volume of the calorimeter cell, XR is the ratio of the total ligand to receptor concentrations at any given point during the titration, and r is defined by

Formula
where n is the number of binding sites per protein molecule, Ka is the association constant, and [M]t is the total protein concentration.

Interaction with TTR
MUPwt and MUPcd were saturated with a 5-fold molar excess of IBMP and incubated for 30 min at room temperature. Unbound ligand was removed and holo proteins were incubated with recombinant TTR (kindly provided by Professor M. J. Saraiva, IBMC, Porto, Portugal) in PBS, pH 7.4, for 30 min at 37°C at a 2:1 molar ratio. Macromolecular mixtures were subjected to size exclusion gel filtration (Superdex-75; Amersham Biosciences Corp., Piscataway, NJ) using an AKTA purifier platform system (Amersham). A 24-ml Superdex-75 column was equilibrated with PBS, pH 7.4, overnight, and 0.4-ml samples were injected (1 mg/ml) and eluted at a flow rate of 0.5 ml/min. Protein peaks were detected by monitoring absorbance at 280 nm, and the elution profiles were compared with those of pure TTR and MUP samples. The molecular mass markers used for calibration were aprotinin (6.5 kDa), cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (67 kDa), and blue dextran (2000 kDa) (Sigma-Aldrich).

Surface plasmon resonance (SPR) binding assays
Where appropriate, recombinant proteins were incubated with ligands at a 5-fold molar excess for 2 h at room temperature. All-trans-retinol (Sigma-Aldrich) was used to obtain holo-RBP or holo-CRBP and IBMP was used to obtain both holo-MUPwt and holo-MUPcd.

1. To confirm that the CD loop of RBP was the main motif responsible for its interaction with the receptor, the chimera MUPcd was compared with RBP for its ability to interact with solubilized membranes from HEK293 cells. SPR was performed using the Biacore 3000 system and NTA sensorchips (Pharmacia Biosensor, Uppsala, Sweden). The sensorchip was activated according to the manufacturer’s instructions, and the buffers used in the microfluidic system were as follows: eluent buffer (0.01 M HEPES, 0.15 M NaCl, 50 µM EDTA, and 0.005% P20, pH 7.4), regeneration buffer (0.01 M HEPES, 0.15 M NaCl, 0.35 M EDTA, and 0.005% P20, pH 8.3). Recombinant holo and apo forms of RBP, MUPwt, and MUPcd were prepared in eluent buffer and injected over the Ni-NTA-activated flow cells at a concentration typically <300 nM. After a contact time of 15 min at a flow rate of 5 µl/min, solubilized diluted membrane preparations (0.2 mg protein/ml) were injected under the same conditions, and binding events were recorded in real time in triplicate. Solubilized erythrocyte "ghost" membranes (0.2 mg protein/ml) were used as a negative control. The sensorchip surface was regenerated with regeneration buffer in a 3-min pulse injection and reactivated with nickel solution (500 µM NiCl2 in eluent buffer) before each subsequent binding experiment.

2. To test for the presence of a receptor for CRBP in placental microvilli membranes, untagged apo-CRBP was coupled onto a CM5 sensorchip by amine coupling chemistry, and SPR was performed using the Biacore 2000 system. Research grade CM5 sensor chips, N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling reagents, ethanolamine, HBS buffer, and glycine-HCl were from Biacore AB. The sensorchip was activated according to the manufacturer’s instructions, and HBS buffer was degassed for 1 h and used as the running buffer. The flow rate was set to 5 µl/min, and the flow cells were activated for 10 min with a mixture of EDC (0.2 M) and NHS (0.05 M). Apo-CRBP was buffer-exchanged in 10 mM sodium acetate (pH 5.2) and then injected at a concentration of 0.5 mg/ml until 1,200 response units were immobilized on three different flow cells, while the fourth flow cell was used as a mock immobilized, blank control. After immobilization, ethanolamine (1 M, pH 8.5) was injected for 10 min to block the remaining activated amine groups. Glycine-HCl (10 mM, pH 2.5) was then used to wash off any nonspecifically bound ligand. Solubilized diluted membrane preparations of placental microvilli, ghosts, or phospholipid vesicles (0.2 mg protein/ml), with or without heat-treatment or addition of metal ions (K+, Ca2+, Mg2+, or Mn2+, 40 mM), were injected at a flow rate of 5 µl/min and allowed a contact time of 15 min, and binding events were recorded in real time in triplicate. Between the injections, the surfaces were regenerated by two 5-µl injections of 10 mM NaOH, after a 10-min wash with the running buffer.

3. To examine whether RBP and CRBP in their holo or apo forms could bind to the same preparation of solubilized HEK293 membranes, RBP was first immobilized onto a Ni-NTA sensorchip (holo-RBP in two flow cells and apo-RBP on the other two) as described in 1 and after an injection of solubilized membrane components (0.2 mg protein/ml), a second injection of either apo/holo-CRBP (untagged) was given at a flow rate of 5 µl/min, allowing a contact time of 15 min. Binding events were recorded in real time in triplicate.

BiaEvaluation software was used for data processing.

Competition binding assays
Binding to placental microvilli membranes was determined by a competitive assay using 35S-labeled MUPcd against a 100-fold molar excess of either unlabeled MUPcd or RBP. The competition assay was adapted from the oil-centrifugation method described in ref. 3 . In the presence or absence of ligand, in a total volume of 200 µl, 0.1 µM freshly labeled protein was incubated with 100 µl of the membrane preparation and a 100-fold molar excess of unlabeled competitor proteins.

Crystallization and data collection
Optimal conditions for crystallization of MUPcd were achieved by hanging drop vapor diffusion in 50 mM CdCl and 100 mM malate buffer (pH 4.7). Drops containing 1 µl of MUPcd (10 mg/ml) and 1 µl of reservoir solution were equilibrated against reservoir solution by vapor diffusion using the hanging drop method. Crystals of space group P43212 grew over a period of 3–7 days. Ligand soaks were performed by adding neat ligand (IBMP) to the reservoir solution to a final concentration of 0.05% (v/v) and allowing the solution to equilibrate with the drop for 24–48 h. After soaking for 1 min in a cryoprotecting solution consisting of reservoir solution with the addition of 20% (v/v) glycerol and 0.05% (v/v) IBMP, crystals were flash-frozen in liquid nitrogen. Data for apo-MUPcd and holo-MUPcd were collected at a Daresbury synchrotron source (Station 14.1) and processed in MOSFLM/SCALA (29) . Structure refinement was accomplished with Refmac (CCP4, 1994) and manual rebuilding in Coot (30) . Crystal coordinates have been deposited in the PDB (accession numbers 2NNE and 2NND).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Substitution of the CD loop of RBP (residues 59–68) into MUP did not alter significantly the ligand-binding ability of the protein, suggesting that the overall protein conformation was retained. ITC measurements on the binding of IBMP to MUPcd and MUPwt at 308 K (Table 1 ) indicated that the enthalpy of binding to MUPcd was more favorable than to that of MUPwt ({Delta}H of {cong}–65 and of {cong}–48, respectively). However, the overall affinity did not change significantly owing to a less favorable change in entropy (Kd values of 0.3 and 0.87 µM for the wild type and the chimera, respectively). This entropy-enthalpy compensation phenomenon is typical of biomolecular associations. None of the native residues removed from MUP (from 58–63) are directly involved in ligand-binding with IBMP as detailed by Bingham et al. (31) when the crystallographic structures for apo- and holo-MUPwt are compared. IBMP is enclosed in a hydrophobic environment formed by the side chains of Phe38, Leu40, Leu42, Ile45, Leu54, Phe56, Met69, Val82, Tyr84, Phe90, Ala103, Leu105, Leu116, and Tyr120.


View this table:
[in this window]
[in a new window]

  		Table 1. Thermodynamic parameters for the binding of IBMP to MUPwt and MUPcd derived from ITC experiments at 308 K

Site-directed mutagenesis studies (20) together with structural data (32) demonstrated that loop EF (residues 89–99) in RBP is a crucial motif for TTR binding, whereas loops AB and CD only contribute additional contacts in macromolecular complex formation. The chromatographic elution profiles of the mixtures of TTR and recombinant MUPwt, MUPcd, and RBP revealed that there was no formation of macromolecular complexes between TTR and MUPwt as the elution profile obtained showed only two isolated peaks at the corresponding molecular weights for each of the individual proteins present (unpublished data).

Having confirmed the functional integrity of MUPcd with respect to ligand binding, the next step was to analyze the behavior of the grafted CD loop in terms of receptor interaction in the presence and absence of a ligand. Binding phenomena between lipocalins immobilized on the sensor surface and components in the solubilized membrane preparations (mobile phase) were analyzed at 37°C. The observed changes in the relative diffraction indices (response units) were recorded as a function of time. All lipocalins were N-terminally his6-tagged and were efficiently immobilized on Ni2+-activated NTA sensorchip surfaces. As an additional control for specific binding, one of the flow cells was activated, subsequently deactivated, and then examined for interaction. Specific binding data for lipocalin-membrane interactions were obtained by subtracting the control flow cell response from those of the immobilized apo/holo lipocalins.

The first set of experiments involved coupling of either apo or holo (ligand-presaturated) forms of MUPwt, MUPcd, and RBP to the same level on different flow cells and then injecting solubilized HEK293 cells membranes over the sensorchip. Figure 1 A shows the high level of response obtained for both holo-MUPcd and -RBP, which contrasted markedly with the lack of significant binding for the apo forms of these two lipocalins. To confirm this result another set of experiments was conducted by flowing a saturable amount of ligand (IBMP or retinol) over the apo proteins immobilized on the sensorchip before the injection of solubilized membranes. The results demonstrated a significant interaction comparable with those obtained for the presaturated holo proteins coupled to the sensorchip.


Figure 1
View larger version (7K):
[in this window]
[in a new window]

  		Figure 1. A) Interaction of apo/holo-lipocalins with solubilized HEK293 membranes. SPR response curves for the interaction of HEK293 cell solubilized membrane preparations flown over immobilized lipocalins: apo- or holo-RBP (a, A), apo- or holo-MUPcd (b, B), and apo- or holo-MUPwt (c, C). The responses were recorded as a function of time and are expressed in response units (RU). Although the levels of binding detected for either the apo or holo forms of MUPwt are almost identical, there is an obvious difference in binding for the apo and holo forms of RBP and MUPcd, with the holo forms showing a highly increased resonance. B) Interaction of holo-lipocalins with HEK293 cells and "ghost" membranes. The responses were recorded as a function of time and are expressed in RU. The increase in resonance, on the injection of HEK293 membranes over the holo-RBP (A) and holo-MUPcd (B) immobilized on the sensorchip, revealed a specific association with membrane components. In contrast, a binding response is not observed on injection of solubilized ghost membranes over holo-RBP (a) and holo-MUPcd (b). The traces obtained with HEK293/ghost membranes for holo-MUPwt were almost identical (C vs. c) and below the level of specific interaction, as shown by the trace of the empty flow cell (d), indicative of an absence of interaction.

To determine whether the significant rise in resonance units was due to the specific binding of holo-lipocalins to membrane receptors or the result of nonspecific interactions with other components in the extract, we heated a pooled fraction of the membrane preparation to 80°C, solubilized it, and analyzed the binding characteristics obtained with the heat-treated preparation. The traces obtained with the heat-treated membranes were identical to the traces obtained with the blank surface, indicating that components in the heated membranes did not interact with the lipocalins (not shown).

When the binding experiment was repeated with solubilized erythrocyte ghosts [this membrane system is not thought to contain the RBP receptor (13 , 33) ], the results showed a response similar to that obtained with heat-treated HEK293 cells membranes or with the blank flow cell, offering further support for a specific receptor-dependent effect (Fig. 1B ).

Again, when MUPwt in either the apo- or holo- forms was immobilized on the sensorchip, no significant specific binding was obtained with solubilized HEK293 membranes as the mobile phase. Taken together, the results obtained indicate that the CD loop of RBP was responsible for the specificity of binding to a membrane receptor present in HEK 293 cells. This finding was evident from the observation that both holo forms of RBP and MUPcd interacted with a component in the solubilized HEK293 cell membranes, but holo-MUPwt could not (Fig. 1A ). In contrast, the apo forms of the lipocalins showed much reduced binding to the solubilized membranes, indicating that the presence of the ligand determines a conformation that is recognized by the receptor. Finally, none of the lipocalins either in the apo or holo forms exhibited interactions with red blood cell membranes, showing that HEK293 cells contain a specific receptor.

These results confirmed that the SPR signal measured with the solubilized membranes from HEK293 cells was due solely to the specific binding of lipocalins with a receptor protein in the presence of a ligand and of loop CD from RBP (grafted or native) and agree with results obtained previously (3 , 22 , 34 , 35) with binding assays using apo and holo preparations of 125I-RBP and placental microvilli membranes. These observations were further confirmed using 35S-labeled MUPcd and placental microvilli membranes (Table 2 ). The raw data showed that the interaction of labeled apo-MUPcd with membranes was nonspecific; however, both IBMP-charged MUPcd and retinol-charged RBP were able to displace radiolabeled holo-MUPcd binding to membranes. Displacement from excess unlabeled holo-RBP was ~81%, whereas displacement for excess unlabeled holo-MUPcd was ~76%.


View this table:
[in this window]
[in a new window]

  		Table 2. Placental microvilli membrane binding assays for MUPcd in the presence or absence of ligand

The crystal structures for the apo and holo forms of MUPcd obtained at 1.6 Å resolution (Table 3 ) reveal only small differences in the backbones of residues 45–46, 53–70, and 156–160 (Fig. 2 A, B). Perhaps significantly, the CD loop region appeared disordered, consistent with high mobility, and, therefore, these residues were deleted from the crystal structures (Fig. 2C, D ). This observation could be attributed to crystal contacts concealing differences that possibly exist in solution. The crystal structures of liganded and unliganded forms of RBP (36) showed that only residues 35 and 36 were rearranged on ligand binding. It is clear from the functional studies, however, that in solution the apo and holo forms must have different conformations, not captured by crystal formation.


View this table:
[in this window]
[in a new window]

  		Table 3. Data collection and refinement statistics for crystal structures for the apo and holo forms of MUPcd obtained at 1.6 Å resolution


Figure 2
View larger version (69K):
[in this window]
[in a new window]

  		Figure 2. Ribbon diagrams of the X-ray crystal structures of holo-MUPcd (A) and apo-MUPcd (B). Highlighted in yellow are the areas of the molecule where residue backbone changes could be seen between the two forms (residues 45–46, 53–70, and 156–160). C, D) Zoomed area between strands C and D shows the electron density at the CD loop region, for the holo (C) and apo (D) forms, respectively. Accession numbers 2NNE and 2NND (PDB).

Earlier, we demonstrated that the transference of retinol from RBP to CRBP was markedly facilitated by the presence of receptor-containing membranes (13) . We examined the possibility that this transference reflected the ability of CRBP to also bind specifically to a receptor in the membrane. Figure 3 illustrates that this is indeed the case, as revealed by SPR experiments similar to the ones described above using a different cell system, this time analyzing the behavior of immobilized CRBP and solubilized placental membranes. The analysis of binding events using membrane preparations from different sources proved that the entity interacting with CRBP was present in placental microvilli but not in erythrocyte ghosts, and that, as seen before, specific binding was greatly diminished by heat treatment. As it was previously reported in RBP binding studies with placental vesicles that divalent ions could have a role in retinol uptake (3) , we examined the effect of the addition of certain divalent cations to placental membranes before injection. Ca2+, in particular, enhanced the binding response to CRBP. The properties of the protein receptor for CRBP in placental microvilli membranes were therefore reminiscent of those for the RBP receptor, most significantly its absence in erythrocyte membranes.


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		Figure 3. Interaction of CRBP with solubilized placental microvilli membranes. The histogram bars summarize the relative binding response of different membrane preparations to immobilized apo-CRBP on a CM5 sensorchip. The normalized response is the binding value at 900 s extracted from each SPR curve, and the error bars represent the SD for three separate experiments. Black bars, the comparison between binding responses of CRBP to phospholipid vesicles, ghost erythrocyte membranes, and placental microvilli membranes; gray bars, the effect of preheating placental microvilli membranes at 80°C compared with unheated preparations; white bars, the effect of preincubation of placental microvilli preparations with 40 mM concentrations of different ionic solutions or just water.

We then investigated whether CRBP in its holo or apo forms could bind to the same entities in solubilized HEK293 membranes as did RBP. Figure 4 illustrates the results. RBP was first attached to the sensorchip, and then solubilized HEK293 membranes were introduced. When CRBP was subsequently applied, another binding event was obtained but only with the apo form of CRBP. Holo-CRBP did not exhibit binding. Neither form of CRBP associated with a control flow cell on the sensorchip containing only RBP.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Figure 4. Interaction of apo/holo-CRBP with solubilized HEK293 membranes. The sensorgrams show the differential interaction of apo- and holo-CRBP (in red and blue, respectively) with solubilized HEK293 cell membranes previously bound to immobilized holo-RBP on two flowcells of a Ni-NTA sensorchip. Three phases are represented on the sensorgrams: 1) immobilization of holo-RBP; 2) interaction of solubilized membranes (putative receptor); and 3) addition of CRBP to RBP-bound solubilized proteins. Only the apo form of CRBP (in red, top panel) showed ability/affinity to bind to the solubilized membranes, suggesting that the absence of the ligand determines a conformation that is recognized by a putative receptor.

The key features of the system are illustrated in Fig. 5 , showing the conformationally sensitive interacting loop and in Fig. 6 , which summarizes the model proposed for the uptake of retinol based on the experimental evidence provided here and earlier.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Figure 5. Conformational hypothesis of the motions of the RBP CD loop that favors interaction with a membrane receptor. Before RBP secretion from the liver, the highly mobile CD loop goes through a series of positional arrangements that allow it in a first instance to become closer to retinol and TTR (holo-RBP) (i, ii, and iii). Once in the serum and upon recognition and binding to the RBP receptor, the CD loop gradually assumes its first position (away from the mouth of the binding pocket), originating other conformational changes that culminate in both TTR release and ligand discharge (apo-RBP). The CD loop is represented in white in the RBP β-barrel. [RETBP_HUMAN: (P02753)] was manipulated using DS Viewer Pro 6.0 to create the hypothetical motions described in the model.


Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		Figure 6. Cartoon representation of the proposed mechanism of RBP/CRBP receptor binding for retinol delivery. Apo-RBP (shown in pale green at the top of the bilayer) complexes with retinol (red) and adopts a holo conformation (dark green). The receptor binding site on the lipocalin becomes accessible and is then able to induce a conformational change in the membrane receptor. This allows transfer of retinol to an intracellular protein such as CRBP (shown beneath the bilayer, in pink) in its apo form.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
By using placental and retina membranes, previous researchers demonstrated that receptors are present which specifically interact with the circulating RBP and induce the release and uptake of retinol from the serum carrier protein (35 , 37) revealing a specific mechanism for retinol release from RBP and subsequent entry into the cell via the receptor. This receptor showed high affinity for the holo form of RBP but not for the apo form (3 , 34) . To further confirm the characteristics of the retinol uptake system, we investigated whether RBP receptor-binding properties could be transferred to another lipocalin, MUP, and whether the same ligand-sensitive conformational effects could be observed. It is apparent from the results presented here that a different lipocalin core, binding a different ligand, is capable of communicating structurally with the grafted RBP loop to effect a conformation that is recognized by the RBP receptor in a ligand-dependent fashion. This finding suggests a conformational coupling mechanism that may be common to many lipocalins and reflects a receptor-dependent biological role.

The binding of RBP to both TTR and the receptor mainly involves different loops near the entrance to the ligand-binding pocket, with TTR via loop EF (residues 89–101) and with the receptor via loop CD (residues 59–68) (20) . The consequences of these interactions also seem to be different. Interaction with TTR has been shown to stabilize the binding of retinol to RBP. In contrast, binding to the cell surface receptor induces the release of retinol from the binding pocket. Amino acids Trp67, Phe96, Leu63, and Leu97 from RBP are flanked by side chains of TTR. Subsequent crystallographic studies on the RBP:TTR complex confirmed these observations. Also, amino acids Leu182 and Leu183 (C terminus) interact with TTR, buried in an hydrophobic patch which includes Leu82 from TTR and Val69 from RBP (32) . The identification of the position of the TTR- and receptor-interacting sites on RBP explains why free RBP rather than the RBP:TTR complex interacts with the membrane receptor (3) . Validating these observations, Melhus et al. (38) using monoclonal antibodies to synthetic peptides identified the same regions on RBP as being involved in TTR and receptor interactions. We suggest that this constitutes an important mechanism for regulating retinol supply to cells.

The data obtained in this study from crystals for apo-and holo-MUPcd showed that with the exception of the grafted CD loop, there was an overall maintenance of the structure compared with that of MUPwt. The analysis of crystals of holo-MUPcd did not disclose the exact conformational change in the liganded form of the protein that allows receptor binding. The only small visible shifts were in the backbone of residues (45–46), (53–70), and (156–160). In both forms, the CD loop appeared to be disordered, which is indicative of a highly flexible region. The CD loop in RBP was also found to be disordered in lower resolution structures of mammalian RBPs determined previously (36 , 39 , 40) . This flexibility, suggested by the reported disorder and localization, has lead to the hypothesis that it could be functionally relevant to retinol binding/release. It should be borne in mind, however, that the liganded form was obtained by soaking the ligand into existing crystals of the apo form, so the CD loop orientation could have been fixed during the crystal growth, and that during the apo/holo transition a transient conformational change is accompanied by the process of retinol binding/release.

With ligand bound, the loop is capable of adopting a high-affinity conformation recognized by the receptor. Both nuclear magnetic resonance (NMR) and crystallographic evidence for MUPwt show that this loop is part of a region that rigidifies when in the holo form (31 , 41 42 43 44) . This finding is also true for MUPcd, being supported by NMR data that indicate differences in backbone chemical shifts of this region between apo- and holo-MUPcd (unpublished observations). In conclusion, we propose that the CD loop in the holo form is less mobile than that in the apo form and when assuming a certain orientation presents a high-affinity conformation that is recognizable by the receptor. The ligand could play an indirect role in the stabilization of loop CD by holding compact the core of MUPcd. So, the present study clearly supports the existence of a reversible conformational state involving a ligand-induced transition that triggers recognition by the membrane receptor.

We therefore put forward a conformational model (Fig. 5) that summarizes the process by which RBP would perform its essential biological role. [i]: In the liver, before secretion, apo-RBPFree possesses a highly mobile CD loop that does not occlude access to the ligand-binding site. After encapsulation of retinol [ii], the loop assumes a position near the mouth of the ligand-binding pocket, holo-RBPFree, helping to lock the ligand in and also becoming part of the contact surface with TTR [amino acids 63 and 67]. The holo-RBP:TTR complex is then secreted into the plasma [iii], thereby reaching the tissues where specific and controlled delivery of retinol is crucial. Receptor recognition at the cell-membrane interface occurs via the CD loop [iv], so the key and so far largely structurally undefined interaction is between this loop and the membrane receptor. At face value, TTR might be expected to hinder this interaction and clearly this appears to be the case from biochemical studies (20) . But, nevertheless, the receptor does access and bind tightly to the CD loop, suggesting a dynamic equilibrium that is reflected in the presence of both TTR-bound and -free forms of RBP. This, in turn [v], may trigger a series of structural motions in the lipocalin, "overwriting" the TTR closed conformation effect. By interacting with the receptor, the CD loop may "unlock" the binding site, provoking deeper conformational changes in RBP, which probably also involve a conformational switch of loop AB [Zanotti et al. (36) proposed that Leu35 controlled the movement of retinol into and out of the RBP ligand-binding pocket] culminating in release/loosening from TTR and discharge of retinol to the receptor. The resultant apo-RBPFree has low affinity for both TTR and the receptor and is rapidly excreted via the kidney [vi]. Figure 5 illustrates hypothetical conformations assumed by the CD loop in the presence or absence of ligand, allowing the interaction/detachment of macromolecular complexes, such as TTR and the receptor.

The exact process by which the receptor facilitates the subsequent transfer of retinol to CRBP, however, has yet to be elucidated. One possibility is that the receptor, either on its own or with associated proteins, might shape the transmembrane mechanism through which retinol is transported into CRBP, with the hydroxyl group leading the way. Such a system would position the retinol in the binding pocket of CRBP in the proper orientation (Fig. 6) . Therefore, retinol undergoes a reorientation when it is delivered by holo-RBP via the plasma membrane receptor to CRBP within the cytoplasm of target cells. Nevertheless, the cavity remains substantially apolar around the ligand. The hydrophobic residues lining the retinol-binding site are in close contact with the bound retinol, such that the vitamin must fit into a rather narrow and highly complementary hydrophobic binding cavity. In the cell, the poorly water-soluble retinol is stored as a retinyl ester derivative of long-chain fatty acids, whose CRBP-dependent synthesis is catalyzed by the enzyme lecithin:retinol acyltransferase (LRAT).

The results presented by van Aalten et al. (45) suggest that the binding site of CRBP to its putative receptor would involve loops CD, EF, and helixII. The earlier study investigating the process by which retinol was transferred from plasma RBP to intracellular apo-CRBP, through the plasma membrane, indicated that the transfer was facilitated by membranes that contained the RBP receptor (13) . In the current work, using SPR and solubilized placental membranes, CRBP exhibited specific Ca2+-dependent binding, which was greatly reduced by heat treatment of membranes. No specific binding was obtained with ghost membranes and phospholipid vesicles, implying that a specific membrane binding site for apo-CRBP might exist on the cytoplasmic side of the membrane.

We next examined whether the specific binding of CRBP was dependent on the apo form of the protein and at the same time whether there was an involvement of RBP. The experiment involved first the application of solubilized HEK293 membranes to the sensorchip containing immobilized holo-RBP and subsequently the introduction of either apo- or holo-CRBP. From the SPR traces shown in Fig. 4 , it can be seen that a receptor element first bound to the immobilized holo-RBP. Subsequent addition of apo- but not holo-CRBP, led to its interaction with the RBP:RBP receptor complex. Because RBP does not interact with CRBP or LRAT, we interpret this result to suggest that a protein component(s) that binds to RBP is also recognized by CRBP. This is the first indication that the mechanism of cellular uptake of retinol from RBP involves not only a specific RBP receptor (as recently validated in ref. 18 ) but also a receptor (the same?) for the retinol recipient CRBP.

These observations are consistent with previous studies (13 , 35) using human placental brush border membranes, in which the holo form of CRBP was ineffective for inducing the release of retinol from the receptor-bound RBP. Clearly, CRBP must also undergo the subtle conformational dynamics exhibited by RBP. Many of the details of this mechanism can now be further elucidated with the identification of the RBP receptor gene (18 , 46) .


   ACKNOWLEDGMENTS
 
We acknowledge the collaboration of Dr. Richard Bingham (University of York, York, UK) for solving the crystal structure of apo and holo MUPcd and also Professor Steve Homans (University of Leeds, Leeds, UK) for performing the ITC binding experiments. This work was supported by grants SFRH/BPD/9435/2002 from Fundação para a Ciência e a Tecnologia, Portugal, and RGBIOC470120, from Biotechnology and Biological Sciences Research Council, UK. We also acknowledge The Welcome Trust for financing the Biacore 2000 and 3000 at Leeds University.

Received for publication August 15, 2007. Accepted for publication October 4, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Newcomer, M. E., Ong, D. E. (2000) Plasma retinol binding protein: structure and function of the prototypic lipocalin. Biochim. Biophys. Acta 1482,57-64[CrossRef][Medline]
  2. Quadro, L., Blaner, W. S., Hamberger, L., Novikoff, P. M., Vogel, S., Piantedosi, R., Gottesman, M. E., Colantuoni, V. (2004) The role of extrahepatic retinol binding protein in the mobilization of retinoid stores. J. Lipid Res. 45,1975-1982[Abstract/Free Full Text]
  3. Sivaprasadarao, A., Findlay, J. B. C. (1988) The interaction of retinol-binding protein with its plasma-membrane receptor. Biochem. J. 255,561-569[Medline]
  4. Davis, J. T., Ong, D. E. (1992) Synthesis and secretion of retinol-binding protein by cultured rat Sertoli cells. Biol. Reprod. 47,528-533[Abstract]
  5. Blomhoff, R., Norum, K. R., Berg, T. (1985) Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells. J. Biol. Chem. 260,13571-13575[Abstract/Free Full Text]
  6. Blaner, W. S., Dixon, J. L., Moriwaki, H., Martino, R. A., Stein, O., Stein, Y., Goodman, D. S. (1987) Studies on the in vivo transfer of retinoids from parenchymal to stellate cells in rat liver. Eur. J. Biochem. 164,301-307[Medline]
  7. Davis, J. T., Ong, D. E. (1995) Retinol processing by the peritubular cell from rat testis. Biol. Reprod. 52,356-364[Abstract]
  8. Yamamoto, M., Drager, U. C., Ong, D. E., McCaffery, P. (1998) Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation. Eur. J. Biochem. 257,344-350[Medline]
  9. Bavik, C. O., Eriksson, U., Allen, R. A., Peterson, P. A. (1991) Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein. J. Biol. Chem. 266,14978-14985[Abstract/Free Full Text]
  10. Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., Eriksson, U. (1993) The retinal pigment epithelial membrane receptor for plasma retinol-binding protein. Isolation and cDNA cloning of the 63-kDa protein. J. Biol. Chem. 268,20540-20546[Abstract/Free Full Text]
  11. Burke, B. J., Redondo, C., Redl, B., Findlay, J. B. C. (2005) Lipocalin receptors: into the spotlight. Akerström, B. Borregaard, N. Flower Edward, D. R. Salier, J. P. eds. Lipocalins Landes Bioscience Austin, Texas, USA.
  12. Redondo, C., Burke, B. J., Findlay, J. B. C. (2006) The retinol-binding protein system: a potential paradigm for steroid-binding globulins?. Horm. Metab. Res. 38,269-278[CrossRef][Medline]
  13. Sundaram, M., Sivaprasadarao, A., DeSousa, M. M., Findlay, J. B. C. (1998) The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. J. Biol. Chem. 273,3336-3342[Abstract/Free Full Text]
  14. Quadro, L., Blaner, W. S., Salchow, D. J., Vogel, S., Piantedosi, R., Gouras, P., Freeman, S., Cosma, M. P., Colantuoni, V., Gottesman, M. E. (1999) Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein. EMBO J. 18,4633-4644[CrossRef][Medline]
  15. Quadro, L., Blaner, W. S., Hamberger, L., Van Gelder, R. N., Vogel, S., Piantedosi, R., Gouras, P., Colantuoni, V., Gottesman, M. E. (2002) Muscle expression of human retinol-binding protein (RBP): suppression of the visual defect of RBP knockout mice. J. Biol. Chem. 277,30191-30197[Abstract/Free Full Text]
  16. Vogel, S., Piantedosi, R., O'Byrne, S. M., Kako, Y., Quadro, L., Gottesman, M. E., Goldberg, I. J., Blaner, W. S. (2002) Retinol-binding protein-deficient mice: biochemical basis for impaired vision. Biochemistry 41,15360-15368[CrossRef][Medline]
  17. Yang, Q., Graham, T. E., Mody, N., Preitner, F., Peroni, O. D., Zabolotny, J. M., Kotani, K., Quadro, L., Kahn, B. B. (2005) Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436,356-362[CrossRef][Medline]
  18. Kawaguchi, R., Yu, J., Honda, J., Hu, J., Whitelegge, J., Ping, P., Wiita, P., Bok, D., Sun, H. (2007) A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315,820-825[Abstract/Free Full Text]
  19. Newcomer, M. E., Jones, T. A., Aqvist, J., Sundelin, J., Eriksson, U., Rask, L., Peterson, P. A. (1984) The three-dimensional structure of retinol-binding protein. EMBO J. 3,1451-1454[Medline]
  20. Sivaprasadarao, A., Findlay, J. B. C. (1994) Structure-function studies on human retinol-binding protein using site-directed mutagenesis. Biochem. J. 300,437-442[Medline]
  21. Sundaram, M., van Aalten, D. M., Findlay, J. B., Sivaprasadarao, A. (2002) The transfer of transthyretin and receptor-binding properties from the plasma retinol-binding protein to the epididymal retinoic acid-binding protein. Biochem. J. 362,265-271[CrossRef][Medline]
  22. Sundaram, M., Sivaprasadarao, A., Findlay, J. B. (1998) Expression and mutagenesis of retinol-binding protein. Methods Mol. Biol. 89,141-153[Medline]
  23. Sivaprasadarao, A., Sundaram, M., Findlay, J. B. (1998) Interactions of retinol-binding protein with transthyretin and its receptor. Methods Mol. Biol. 89,155-163[Medline]
  24. Boudjelal, M., Sivaprasadarao, A., Findlay, J. B. C. (1996) Membrane receptor for odour-binding proteins. Biochem. J. 317,23-27[Medline]
  25. Cavaggioni, A., Findlay, J. B., Tirindelli, R. (1990) Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine-binding protein of calf. Comp. Biochem. Physiol. B 96,513-520[CrossRef][Medline]
  26. Dodge, J. T., Mitchell, C., Hanahan, D. J. (1963) The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys. 100,119-130[CrossRef][Medline]
  27. Wiseman, T., Williston, S., Brandts, J. F., Lin, L. N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179,131-137[CrossRef][Medline]
  28. Turnbull, W. B., Daranas, A. H. (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry?. J. Am. Chem. Soc. 125,14859-14866[CrossRef][Medline]
  29. CCP4, Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50,760-763[CrossRef][Medline]
  30. Emsley, P., Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60,2126-2132[CrossRef][Medline]
  31. Bingham, R. J., Findlay, J. B., Hsieh, S. Y., Kalverda, A. P., Kjellberg, A., Perazzolo, C., Phillips, S. E., Seshadri, K., Trinh, C. H., Turnbull, W. B., Bodenhausen, G., Homans, S. W. (2004) Thermodynamics of binding of 2-methoxy-3-isopropylpyrazine and 2-methoxy-3-isobutylpyrazine to the major urinary protein. J. Am. Chem. Soc. 126,1675-1681[CrossRef][Medline]
  32. Naylor, H. M., Newcomer, M. E. (1999) The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP. Biochemistry 38,2647-2653[CrossRef][Medline]
  33. Chen, C. C., Heller, J. (1977) Uptake of retinol and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells. J. Biol. Chem. 252,5216-5221[Free Full Text]
  34. Sivaprasadarao, A., Findlay, J. B. C. (1988) The mechanism of uptake of retinol by plasma-membrane vesicles. Biochem. J. 255,571-579[Medline]
  35. Sivaprasadarao, A., Boudjelal, M., Findlay, J. B. (1993) Lipocalin structure and function. Biochem. Soc. Trans. 1993 21(Pt 3),619-622[Medline]
  36. Zanotti, G., Berni, R., Monaco, H. L. (1993) Crystal structure of liganded and unliganded forms of bovine plasma retinol-binding protein. J. Biol. Chem. 268,10728-10738[Abstract/Free Full Text]
  37. Bavik, C. O., Busch, C., Eriksson, U. (1992) Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium. J. Biol. Chem. 267,23035-23042[Abstract/Free Full Text]
  38. Melhus, H., Bavik, C. O., Rask, L., Peterson, P. A., Eriksson, U. (1995) Epitope mapping of a monoclonal antibody that blocks the binding of retinol-binding protein to its receptor. Biochem. Biophys. Res. Commun. 210,105-112[CrossRef][Medline]
  39. Cowan, S. W., Newcomer, M. E., Jones, T. A. (1990) Crystallographic refinement of human serum retinol binding protein at 2A resolution. Proteins 8,44-61[CrossRef][Medline]
  40. Zanotti, G., Panzalorto, M., Marcato, A., Malpeli, G., Folli, C., Berni, R. (1998) Structure of pig plasma retinol-binding protein at 1.65 A resolution. Acta Crystallogr. D Biol. Crystallogr. 54(Pt 5),1049-1052[CrossRef][Medline]
  41. Timm, D. E., Baker, L. J., Mueller, H., Zidek, L., Novotny, M. V. (2001) Structural basis of pheromone binding to mouse major urinary protein (MUP-I). Protein Sci. 10,997-1004[CrossRef][Medline]
  42. Perazzolo, C., Wist, J., Loth, K., Poggi, L., Homans, S., Bodenhausen, G. (2005) Effects of protein-pheromone complexation on correlated chemical shift modulations. J. Biomol. NMR 33,233-242[CrossRef][Medline]
  43. Barratt, E., Bingham, R. J., Warner, D. J., Laughton, C. A., Phillips, S. E., Homans, S. W. (2005) Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc. 127,11827-11834[CrossRef][Medline]
  44. Perazzolo, C., Verde, M., Homans, S. W., Bodenhausen, G. (2007) Evidence of chemical exchange in recombinant major urinary protein and quenching thereof upon pheromone binding. J. Biomol. NMR 38,3-9[CrossRef][Medline]
  45. van Aalten, D. M., Findlay, J. B., Amadei, A., Berendsen, H. J. (1995) Essential dynamics of the cellular retinol-binding protein—evidence for ligand-induced conformational changes. Protein Eng. 8,1129-1135[Abstract/Free Full Text]
  46. Moise, A. R., Noy, N., Palczewski, K., Blaner, W. S. (2007) Delivery of retinoid-based therapies to target tissues. Biochemistry 46,4449-4458[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.07-8939comv1
22/4/1043    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Redondo, C.
Right arrow Articles by Findlay, J. B. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Redondo, C.
Right arrow Articles by Findlay, J. B. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS