©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Chicken Oocyte Receptor for Yolk Precursors as a Model for Studying the Action of Receptor-associated Protein and Lactoferrin (*)

(Received for publication, March 9, 1995)

Thomas Hiesberger (1) Marcela Hermann (1) Linda Jacobsen (§) Sabine Novak (1) Regina A. Hodits (2) Hideaki Bujo (1) Melinda Meilinger (3) Manfred Httinger (3) Wolfgang J. Schneider (1) Johannes Nimpf (1)(¶)

From the  (1)Departments of Molecular Genetics and (2)Medical Biochemistry, Biocenter and University of Vienna and (3)Department of Medical Chemistry, University of Vienna, A-1030 Vienna, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Receptor-associated protein (RAP) was originally described as a 39-kDa intracellular protein copurifying with mammalian low density lipoprotein (LDL) receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR). RAP has a high affinity for LRP/alpha(2)MR and interferes with the receptor's ability to bind a variety of ligands. The laying hen expresses, in a tissue-specific manner, at least four different proteins which belong to the same family of receptors as LRP/alpha(2)MR. Here we show that the chicken also produces RAP, so far thought to be expressed only in mammals. Studies on the interaction of recombinant human RAP with the LDL receptor family in the chicken revealed that RAP binds with high affinity to the abundant oocyte receptor for yolk precursors (OVR) as well as to the somatic cell-specific LRP/alpha(2)MR. Significantly, RAP interacts with a lower affinity with the LDL receptor, but does not bind to the oocyte-specific form of LRP. Binding of RAP to OVR inhibits the interaction of the receptor with all known physiological ligands, i.e. the yolk precursors very low density lipoprotein, vitellogenin, and alpha(2)-macroglobulin. In COS cells transfected with OVR, RAP is internalized and degraded in a concentration-dependent and saturable manner. Lactoferrin, another protein with a high affinity for mammalian LRP/alpha(2)MR, also binds to OVR and abolishes its interaction with yolk precursors. Cross-competition experiments show that RAP and lactoferrin recognize sites different from those involved in yolk precursor binding. The availability of pure OVR and LDLR enabled us to determine kinetic parameters for the binding of RAP and lactoferrin to these receptors by surface plasmon resonance. Taken together, our results strongly suggest that chicken OVR, which is easily accessible and highly abundant in growing oocytes, represents a superior system for studying mechanistic and structural aspects of the interaction of ligands and modulating proteins with members of the LDL receptor gene family.


INTRODUCTION

We have characterized at the molecular and biochemical level the chicken oocyte receptor (OVR) (^1)for the yolk precursors, very low density lipoprotein (VLDL) and vitellogenin (VTG) (1) . This receptor is the avian homologue of the mammalian so-called VLDL receptor which was recently cloned from rabbit(2) , man(3, 4, 5, 6) , mouse(7, 8) , and rat(9) . The structural hallmark of VLDL receptors is a cluster of 8 cysteine-rich binding repeats (2) highly homologous to the 7 binding repeats found in the LDL receptor(10) . The mammalian VLDL receptor displays high affinity for apoE-containing lipoproteins, especially VLDL(2) , but its true physiological function is not understood. In contrast, the chicken oocyte receptor is known to be the key player in normal oocyte development as demonstrated by the receptor-deficient genetic model, the restricted ovulator hen(11, 12) . The absence of expression of functional OVR in oocytes of restricted ovulator hens (13) is the cause for the failure to lay eggs (i.e. female sterility) associated with severe hyperlipidemia. Normally, OVR is responsible for the rapid uptake of the major yolk precursors into growing oocytes (for review, see Refs. 14 and 15). These precursors, which make up about 50% of the total weight of the egg yolk, are VLDL and VTG. Both macromolecules bind specifically and with high affinity to the 95-kDa OVR(16, 17) localized in coated pits in the plasma membrane of growing oocytes (18) . In addition, OVR was found to be the receptor for certain minor yolk components, such as riboflavin-binding protein (19) and alpha(2)-macroglobulin (alpha(2)M)(20) .

The best characterized member and the prototype of the LDL receptor family is the LDL receptor itself, which contains all modules commonly found in members of this receptor family(10) : (i) the above mentioned ``binding repeats,'' complement-type domains consisting of 40 residues displaying a triple disulfide bond-stabilized, negatively charged surface (certain head-to-tail combinations of these repeats are believed to specify ligand interactions); (ii) epidermal growth factor precursor-type repeats, also containing 6 cysteines each; (iii) modules of 50 residues with a consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD); and (iv), in the cytoplasmic region, signals for receptor internalization via coated pits, containing the consensus tetrapeptide Asn-Pro-Xaa-Tyr (NPXY).

The LDL receptor-related protein is another well studied member of this gene family. This giant receptor contains 4 clusters of 2-11 complement type binding repeats each and can bind, at least in vitro, an ever growing number of different ligands including alpha(2)M-proteinase complexes (21, 22) (this receptor was therefore named LDL receptor-related protein/alpha(2)M receptor; LRP/alpha(2)MR), apoE(23) , apoE-enriched lipoproteins(24, 25) , lipoprotein lipase(26, 27, 28) , plasminogen activators and/or complexes with their respective endogenous inhibitors (29, 30) , Pseudomonas exotoxin A(31, 32) , and rhinoviruses of the minor group(33) .

A 39-kDa protein (receptor-associated protein, RAP) was described which copurifies with LRP/alpha(2)MR from liver and placenta (34, 35, 36) . RAP is an intracellular protein with high affinity for LRP/alpha(2)MR and the ability to compete for binding of most known ligands to the receptor(21, 30, 31, 37, 38) . Furthermore, following binding to the receptor, RAP is rapidly internalized and degraded, as shown with human monocytes(39) , cultured fibroblasts (40) , and transfused rat livers(41) . Although conflicting results have been obtained in the assessment of the cellular localization of RAP (42, 43, 44) , the majority of the protein seems to be located intracellularly(45) , and, under normal conditions, RAP could not be detected in plasma(34) . Herz (40) postulated that RAP might modulate LRP/alpha(2)MR activity in vivo by associating with LRP/alpha(2)MR in recycling vesicles before the receptor reaches the plasma membrane. RAP was successfully used in vivo to demonstrate LRP/alpha(2)MR's function as a backup mechanism for the clearance of chylomicron remnants in rodents(41, 46) . Recently it was shown that RAP also binds to the human VLDL receptor and antagonizes the binding of VLDL to this receptor(47) .

LRP/alpha(2)MR also binds lactoferrin, possibly by a tetrabasic sequence motive (RXXRKR) similar to the putative binding site of apoE(48) . Lactoferrin is an iron-binding protein and belongs to a family of related proteins including transferrin and ovotransferrin(49) . Its actual physiological function is not clear yet. Present in high concentrations in milk, it was suggested that it may act as a primary defence barrier against microbial infection(50) . In a recent publication it was shown that lactoferrin interacts with DNA, exerting a distinct sequence specificity leading to direct transcriptional activation(51) . However, lactoferrin was used in vivo as a potent inhibitor of the clearance of chylomicron remnants and beta-migrating very low density lipoprotein(52, 53) . Interestingly, uptake of activated alpha(2)M was not blocked by lactoferrin in this system. Using lactoferrin in ligand blots with rat liver and kidney extracts, its binding to LRP/alpha(2)MR and gp330 (megalin), another member of the LDL receptor family(54) , was directly demonstrated(55, 56) . Degradation of I-alpha(2)M in LDL receptor-negative fibroblasts was strongly inhibited by RAP but not by lactoferrin(56) .

In the present study we demonstrate for the first time expression of RAP in a non-mammalian system and that both RAP and lactoferrin bind to the chicken oocyte receptor for yolk precursor uptake. Both ligands effectively displace all of the currently identified ligands from the receptor, i.e. VLDL, VTG, and activated and native alpha(2)M. This, for lactoferrin, is in sharp contrast to its interaction with LRP/alpha(2)MR.


EXPERIMENTAL PROCEDURES

Animals and Diets

White Leghorn laying hens were purchased from Heindl (Vienna) and maintained as described elsewhere(57) . Roosters (20-30 weeks old) were treated with 17alpha-ethinylestradiol dissolved in propylene glycol, by injecting 10 mg/kg of body weight into the breast muscle. After 72 h, blood was collected from the jugular vein and mixed with the following additives to result in the indicated final concentrations: 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, 0.1 µM aprotinin.

Preparation of Oocyte Membranes

Oocyte membranes were prepared from previtellogenic follicles (4-6-mm diameter) excised from mature laying hens and extracted with 1% Triton X-100 as described previously(57) .

Preparation of Antibodies

Polyclonal antibodies against OVR were described previously(1) . A sequence-specific antibody against chicken somatic LRP/alpha(2)MR was obtained using a synthetic peptide corresponding to the 14 carboxyl-terminal amino acids of the heavy chain of chicken LRP/alpha(2)MR (residues 3928-3942) (58) . The peptide was coupled to keyhole limpet hemocyanin (59) and used for immunization of New Zealand White rabbits as described elsewhere(60) . IgG fractions were purified from sera on protein A-Sepharose CL-4B matrix (Pharmacia Biotech Inc.) according to Beisiegel et al.(61) . Immune-purified anti-human RAP antibody was a generous gift of Dr. D. K. Strickland (American Red Cross, Rockville, MD).

Preparation and Radiolabeling of Ligands

Very low density lipoprotein was prepared from plasma of estrogen-treated roosters by sequential ultracentrifugation according to George et al.(57) . LDL was prepared from plasma of laying hens according to Hayashi et al.(62) . Vitellogenin was purified from estrogenized rooster plasma by ion exchange chromatography (DEAE-cellulose) as previously described(63) . Both lipoproteins were labeled with I to a specific activity of 250-400 cpm/ng using the iodine monochloride method as described previously(64) . alpha(2)M was purified from citrated plasma of laying hens essentially according to Sottrup-Jensen et al.(65) with minor modifications as described previously(20) . alpha(2)M-trypsin complexes (alpha(2)M*) were prepared by incubating native alpha(2)M with a 15-fold molar excess of trypsin for 1 min followed by addition of the same amount of soybean trypsin inhibitor. Native alpha(2)M was radioiodinated using the lactoperoxidase method (66) to a specific activity of 900-1100 cpm/ng. Labeled alpha(2)M-trypsin complexes were produced by reacting labeled native alpha(2)M with trypsin as described above. Recombinant RAP was produced as a glutathione S-transferase (GST) fusion protein using a PGEX 2T-derived (Pharmacia) expression plasmid in DH5alpha bacteria(37) . Purified RAP-GST was iodinated using chloramine T according to Sambrook et al.(68) . Bovine lactoferrin (Serva, Austria) was iodinated by the IODO-BEAD method according to the manufacturer's specification (Pierce). Typically, 0.1 mg of a 15 µM solution of lactoferrin in TBS (50 mM Tris, 150 mM NaCl, pH 8) was iodinated with 0.1 mCi of NaI and one IODO-BEAD to a specific activity of 1000-1500 cpm/ng. Labeled ligands were separated from free I by passing them over a PD10 column (Pharmacia). After extensive dialysis against TBS, 0.1 mM EDTA, they were stored at 4 °C for up to 2 weeks.

Electrophoresis and Western and Ligand Blotting

One-dimensional SDS-PAGE under nonreducing conditions was performed according to Laemmli (69) on 4.5-18% or 4.5-12% gradient slab gels at 180 V for 60 min using the minigel system from Bio-Rad. Transfer to nitrocellulose and Western and ligand blotting was performed as described previously(20) .

Cell Culture Experiments

Chicken embryo fibroblasts were obtained and cultured by methods that have been previously described (62) . For suppression of the LDL receptor, fibroblast monolayers were incubated with fetal bovine serum supplemented with 25-OH-cholesterol (4 µg/ml). To stimulate expression of the LDL receptor, fibroblast monolayers were incubated for 24 h in medium containing 10% lipoprotein-deficient serum supplemented with mevinolin (2 µg/ml). For ligand and Western blotting experiments cell monolayers were processed as described elsewhere(62) .

To determine cell binding of RAP, monolayers of COS-7 cells transiently transfected with pCDMCVR-1, a plasmid-carrying, full-length cDNA for OVR(1) , and control cells (transfected with pCDM8), were incubated for 3 h at 4 °C in standard medium containing 2 mg/ml bovine serum albumin and the concentrations of radioiodinated and unlabeled ligands as indicated in the figure legends. The medium was then removed, and the monolayers were carefully washed to remove unbound ligand as described previously(62) . Cell-associated radioactivity was determined by a -counter (Cobra II, Packard Instr.) following solubilization of the cells in 1 ml of 0.1 N NaOH. Assays for proteolytic degradation of I-labeled ligands in monolayers of cultured cells were determined according to the standard protocol for low density lipoprotein(70) . Solubilization of the cells, SDS-PAGE, electrophoretic transfer, and ligand blotting were performed as described above.

Purification of OVR and Bovine LDL Receptor

OVR was purified in a two-step procedure applying affinity chromatography on RAP-Sepharose and ion exchange chromatography on a Mono-Q column (fast protein liquid chromatography; Pharmacia). GST-RAP was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions (5 mg/ml column material). Twenty ml of oocyte membrane extract (prepared in 125 mM Tris/maleate, pH 6, 1 mM CaCl(2), 160 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 30 mM CHAPS) were applied onto a column containing 10 ml of GST-RAP-Sepharose. The flow-through was recycled three times over the same column. The column was washed with 250 ml of buffer A (50 mM Tris/HCl, pH 8, 2 mM CaCl(2), 30 mM CHAPS). Bound protein was eluted with a linear gradient (13.5 ml) of buffer A containing 0-1 M NH(4)OH. Fractions containing the protein were directly applied onto a Mono-Q column equilibrated with buffer B (50 mM Tris/HCl, pH 8, 2 mM CaCl(2), 16 mM CHAPS). After extensive washing (until base-line absorption at 280 nm was reached) with buffer B, OVR was eluted with a linear gradient (22 ml) of buffer B containing 0-400 mM NaCl.

LDL receptor was purified from bovine adrenal cortex by DEAE-cellulose and affinity chromatography on LDL-Sepharose as described elsewhere (71) .

Determination of Binding Kinetics by Surface Plasmon Resonance

Surface plasmon resonance experiments were performed on a BIAcore instrument (Pharmacia Biosensor AB). Purified OVR and bovine LDL receptor were coupled to CM5 sensor chips using the amine coupling kit (Pharmacia Biosensor AB) according to Johnsson et al.(72) . Receptor preparations (450 ng) in 10 mM NaAc, pH 4.0 (for OVR) or pH 3.0 (for LDL receptor), were used and yielded 1000 response units of immobilized protein for OVR and 250 response units for LDL receptor, respectively. All determinations were performed at 25 °C in 10 mM HEPES, 150 mM NaCl, 2 mM CaCl(2), 0.05% BIAcore surfactant P20, pH 7.4, with a constant flow rate of 5 µl/min and an injected sample volume of 35 µl for RAP and lactoferrin, respectively. RAP was used at concentrations between 7 10M and 2 10M. Lactoferrin was used at concentrations between 3 10M and 1 10M. Two consecutive 4-µl injections of 10 mM HCl were used for regeneration. The calculation of the off and on rates was performed with the BIAevaluation Program 2.0 (Pharmacia Biosensor) assuming first order kinetics.

Other Procedures

The protein content of samples containing Triton X-100 or lipoproteins were measured by a modified Lowry procedure(73) . Other samples were measured according to the original method of Lowry et al.(74) .


RESULTS

To investigate the interaction of RAP with members of the chicken LDL receptor gene family, we first compared membrane extracts derived from the liver and ovarian follicles. The follicle expresses at least three distinct members of this family which can be visualized by Western and ligand blotting of crude membrane extracts. As shown in Fig. 1A (lane 4), I-GST-RAP binds to two distinct proteins present in crude follicular membrane extracts with relative molecular masses of 95 kDa and approximately 500 kDa, respectively. In contrast to the follicular extracts, liver membrane extracts contain the higher molecular weight band only (lane 5). Both bands are specific for the RAP portion of the fusion protein, since I-GST did not react with any proteins in these extracts (data not shown). To further characterize the bands visualized with GST-RAP, we performed Western blots with a panel of specific antibodies against chicken LDL receptor family members. Lane 1 of Fig. 1A shows OVR in membranes of ovarian follicles visualized with a sequence-specific polyclonal antibody(1) . In lane 2, we used a polyclonal antibody against purified OVR which cross-reacts with oocyte-specific LRP (molecular mass of approximately 380 kDa) present in ovarian membranes(75) . In addition, binding of RAP-GST to OVR was proven on a strip containing purified OVR only (lane 3). This experiment clearly shows that (i) the smaller RAP-binding protein in follicular membranes is identical with OVR and (ii) that the oocyte-specific LRP does not bind RAP. To characterize the 500-kDa RAP-binding protein present in follicular and liver membranes, we used a new antibody prepared against the carboxyl terminus of the heavy chain (515 kDa) of the chicken somatic LRP/alpha(2)MR (58) as described under ``Experimental Procedures.'' In lane 6, this antibody visualizes the somatic LRP/alpha(2)MR-515 in liver membranes, which comigrates with the RAP-binding protein in lanes 4 and 5.


Figure 1: Western and ligand blots of purified OVR and membrane extracts prepared from chicken tissues and from cells transfected with OVR expression plasmid. Triton X-100 membrane extracts from chicken liver and follicles and COS-7 cells were prepared, electrophoresis performed under nonreducing conditions on 4.5-18% SDS-polyacrylamide gradient gels, and proteins were electrophoretically transferred to nitrocellulose as described under ``Experimental Procedures.'' The positions of marker proteins are shown. A, lanes 1, 2, and 4, Triton X-100 extracts of chicken follicle membranes (5 µg of protein/lane); lane 3, 200 ng of purified OVR; lanes 5 and 6, Triton X-100 extracts of chicken liver membranes (15 µg of protein/lane). Nitrocellulose membranes were incubated with a sequence-specific antibody against OVR (10 µg/ml) (lane 1); a polyclonal antibody against OVR and oocyte-specific LRP (1 µg/ml) (lane 2); I-RAP-GST (0.5 µg/ml; specific activity, 1 10^3 cpm/ng, lanes 3-5); and a polyclonal antibody against somatic LRP-515 (10 µg/ml) (lane 6). Bound antibodies were visualized with protein A-HRP and the chemiluminescence system described under ``Experimental Procedures.'' Exposure time was 1 min for lanes 1 and 2, 3 min for lane 6, and 24 h for lanes 3-5. B, lanes 1, 3, 5, Triton X-100 extracts of chicken follicle membranes (5 µg of protein/lane); lanes 2, 4, 6, Triton X-100 extracts of chicken liver membranes (15 µg of protein/lane). Nitrocellulose membranes were incubated with I-RAP-GST (0.5 µg/ml; specific activity, 1 10^3 cpm/ng) without further additions (lanes 1 and 2), in the presence of 0.5 mg/ml unlabeled RAP-GST (lanes 3 and 4) and in the presence of 20 mM EDTA (lanes 5 and 6). Exposure time was 24 h. C, COS-7 cells were transiently transfected with the OVR expression plasmid pCDMCVR-1 (lanes 1 and 3) or with a control plasmid (lanes 2 and 4) and processed for ligand and Western blotting following SDS-PAGE under nonreducing conditions as described under ``Experimental Procedures.'' Nitrocellulose membranes were incubated with I-RAP-GST (0.5 µg/ml; specific activity, 1 10^3 cpm/ng, lanes 3 and 4), or with a sequence-specific antibody against OVR (10 µg/ml, lanes 3 and 4). The ligand blot was exposed for 24 h. The Western blot was processed as described in A, and exposure time was 2 min.



To assess the specificity of RAP binding, we performed ligand blots in the presence of a 500-fold molar excess of unlabeled GST-RAP. As shown in Fig. 1B, unlabeled GST-RAP (lanes 3 and 4) strongly interferes with the binding of the labeled ligand (lanes 1 and 2) to OVR as well as to somatic LRP/alpha(2)MR as assayed in follicle (lanes 1 and 3) and liver (lanes 2 and 4) membrane extracts. Again, the same amount of unlabeled GST did not block binding of GST-RAP to the two proteins (data not shown). Addition of 40 mM EDTA to the ligand blot buffer completely abolished the interaction of GST-RAP with the receptors (lanes 5 and 6), demonstrating the dependence of the binding on divalent cations.

Recently we could demonstrate that transfection of COS-7 cells with a full-length cDNA for OVR leads to the functional expression of OVR, as determined by binding of VLDL and VTG to these cells(1) . Here we used this experimental system to show that the 95-kDa RAP-binding protein in follicular membranes is indeed OVR. As shown in Fig. 1C, both the polyclonal antibody against purified OVR and I-GST-RAP bind to OVR expressed by cells transfected with the full-length cDNA for OVR (lanes 1 and 3). In contrast, mock transfected cells (lane 4) do not express any immunoreactive protein. The small amount of residual binding of RAP to a band of the same or similar size as the exogenous OVR (lane 2) might be due to the endogenous simian VLDL receptor, which is not recognized by the chicken-specific antibody (lane 4)(1) .

To assess internalization competence of OVR toward RAP, we used the transfected COS-7 cells to study OVR-mediated binding and degradation of RAP. As shown in Fig. 2A, cells expressing OVR specifically bind labeled RAP-GST. Degradation studies using transfected and control cells demonstrated that OVR-bound RAP is intracellularly degraded (Fig. 2B). Both binding and degradation showed high affinity and saturation kinetics; at 20 µg/ml RAP, maximal degradation amounted to 600 ng of I-RAP-GST/mg of cell protein in 5 h. Control experiments using conditioned media from transfected and control cells showed that extracellular degradation of RAP amounted to less than 1% of the total activity and does not exceed values obtained when RAP was incubated with fresh medium without cells for 5 h at 37 C°.


Figure 2: Binding and degradation of RAP-GST by COS-7 cells expressing OVR. COS-7 cells were transiently transfected with the expression plasmid for OVR or the control plasmid as described under ``Experimental Procedures.'' A, 48 h after transfection, cell monolayers were incubated with the indicated concentrations of labeled RAP-GST for 3 h at 4 °C. Bound ligand was quantitated as described under ``Experimental Procedures.'' Values were corrected for no-cell blanks and OVR-mediated binding was calculated by subtracting the values obtained for control cells from those obtained for OVR-expressing cells. Each value represents the average of triplicate determinations. B, 48 h after transfection, cell monolayers received 2 ml of standard medium containing the indicated concentrations of I-RAP-GST. After 5 h of incubation, degradation products secreted into the medium were measured as amount of TCA-soluble radioactivity recovered from the cell supernatant. No-cell blanks were subtracted from the values obtained for OVR-expressing and control cells. Specific, OVR-mediated degradation was calculated by substracting the values for control cells from those obtained for OVR-expressing cells. Each value represents the average of triplicate determinations.



The finding that RAP, which was originally described as ligand for LRP/alpha(2)MR, binds to OVR prompted us to investigate whether lactoferrin, another ligand of LRP/alpha(2)MR(56) , is recognized by the same receptor. As demonstrated in Fig. 3, radiolabeled lactoferrin binds to purified OVR (lane 1) and OVR present in crude follicle membrane extracts (lane 2). As expected, the binding is inhibited by an excess of unlabeled lactoferrin (lane 3). Furthermore, lactoferrin binding is inhibited by RAP-GST (lane 4) and is Ca-dependent (lane 5).


Figure 3: Ligand blot of purified OVR and chicken follicle membrane extracts with lactoferrin. Purified OVR (lane 1, 200 ng) and Triton X-100 membrane extracts from chicken follicles (lanes 2-5, 5 µg of protein/lane) were electrophoretically separated on 4.5-12% SDS-polyacrylamide gradient gels under nonreducing conditions. Proteins were transferred to nitrocellulose, and the membranes were incubated with I-lactoferrin (1 µg/ml, 1 10^3 cpm/ng) in standard ligand blot buffer with the following additions: lanes 1 and 2, none; lane 3, unlabeled lactoferrin (100 µg/ml); lane 4, unlabeled RAP-GST (100 µg/ml); lane 5, 20 mM EDTA. The position of marker proteins are shown. Exposure time was 48 h.



Having demonstrated that RAP and lactoferrin bind to chicken OVR, we tested whether these ligands would interfere with binding of yolk precursors to their common receptor. For the first set of experiments (Fig. 4A) we used labeled RAP-GST as a ligand in the presence of a 1000-fold molar excess of RAP or unlabeled yolk precursors. As shown in lane 1, RAP binds to OVR and somatic LRP (see also Fig. 1A). Binding is completely abolished by an excess of unlabeled RAP (lane 2). VLDL (lane 3) does not interfere with RAP binding, whereas VTG (lane 4) markedly reduces the signal produced by labeled RAP. Native (lane 5) and trypsin-treated (lane 6) alpha(2)M do not significantly interfere with the receptor's ability to bind RAP. Lactoferrin (lane 7), however, was almost as potent a competitor for RAP binding as RAP itself.


Figure 4: Cross-competition of RAP and lactoferrin with VLDL, VTG, and alpha(2)M. Follicle membrane Triton X-100 extract (10 µg of protein/lane) was subjected to electrophoresis on a 4.5-12% SDS-gradient PAGE under nonreducing conditions and transferred to nitrocellulose, and ligand blotting was performed as described under ``Experimental Procedures.'' A, membranes were incubated with I-RAP-GST (0.5 µg/ml, 1 10^3 cpm/ng) with the following additions: lane 1, none; lane 2, unlabeled RAP-GST (0.5 mg/ml); lane 3, VLDL (1 mg/ml); lane 4, VTG (0.5 mg/ml); lane 5, alpha(2)M (0.5 mg/ml); lane 6, alpha(2)M* (0.5 mg/ml); and lane 7, lactoferrin (0.5 mg/ml). Exposure time was 24 h. B, membranes were incubated with I-VLDL (5 µg/ml, 250 cpm/ng) with the following additions: lane 1, none; lane 2, unlabeled VLDL (1 mg/ml); lane 3, RAP-GST (2.5 µg/ml); lane 4, RAP-GST (25 µg/ml); lane 5, RAP-GST (250 µg/ml); and lane 6, lactoferrin (250 µg/ml). Exposure time was 24 h. C, membranes were incubated with I-VTG (2 µg/ml, 300 cpm/ng) with the following additions: lane 1, none; lane 2, unlabeled VTG (0.5 mg/ml); lane 3, RAP-GST (2.5 µg/ml); lane 4, RAP-GST (25 µg/ml); lane 5, RAP-GST (250 µg/ml); and lane 6, lactoferrin (250 µg/ml). Exposure time was 24 h. D and E, membranes were incubated with native I-alpha(2)M (7 ng/ml, 13 10^3 cpm/ng) for Panel D and with I-alpha(2)M* (7 ng/ml, 12 10^3 cpm/ng) for Panel E with the following additions: lanes 1, none; lanes 2, RAP-GST (250 µg/ml); and lanes 3, lactoferrin (250 µg/ml). Exposure time was 2 d.



We then used labeled yolk precursors and evaluated the cross-competition with RAP and lactoferrin, respectively. As expected, I-VLDL-binding (Fig. 4B, lane 1) and I-VTG-binding (Fig. 4C, lane 1) are both abolished by a 1000-fold molar excess of the respective unlabeled ligand (lanes 2). RAP-GST competed for both ligands in a concentration-dependent manner (lanes 3-5), reducing the signals produced by I-VLDL and I-VTG to background levels at a 1000-fold molar excess in the ligand blot incubation medium. Under these conditions, the effect of lactoferrin (1000-fold molar excess) was indistinguishable from that of RAP, as shown in Panels B and C (lanes 6), completely displacing both natural ligands from the receptor.

As recently demonstrated, OVR also binds native and trypsin-treated alpha(2)M(20) . To further characterize this binding, we performed competition experiments with RAP and lactoferrin. As demonstrated in Fig. 4D for I-alpha(2)M and in Fig. 4E for I-alpha(2)M*, addition of RAP-GST competes for the binding of both ligands (lanes 2). Lactoferrin also interferes with the binding of I-alpha(2)M and I-alpha(2)M* (Fig. 4, D and E, lane 3), but to a somewhat smaller extent than RAP under these conditions.

Although it was originally reported that RAP does not bind detectably to the LDL receptor(37) , recent results obtained by injecting large amounts of RAP into rats (41) and binding studies on human fibroblasts (76) suggested that RAP might interact with the LDL receptor as well as with LRP/alpha(2)MR. We therefore directly investigated the interaction of RAP and lactoferrin with the chicken LDL receptor. We used primary chicken embryo fibroblasts in which expression of the LDL receptor was maximally stimulated or suppressed(62) . In induced cells, labeled chicken LDL strongly visualized the chicken LDL receptor (M(r) 130,000) in detergent extracts (Fig. 5A, lane 1). This band is absent in membrane extracts from cells in which LDL receptor expression had been suppressed (lane 2). As demonstrated in lane 3, labeled RAP binds to the chicken LDL receptor. The signal produced by RAP is indeed produced by the LDL receptor, since it was undetectable in membrane extracts from cells grown under suppressing conditions (lane 4). Under the same conditions, however, binding of lactoferrin to the chicken LDL receptor (induced fibroblasts) in ligand blots could not be detected (data not shown). To analyze whether the interaction of RAP is unique for the chicken LDL receptor, we performed similar ligand binding experiments using purified bovine LDL receptor. As shown in Fig. 5B, the bovine LDL receptor also binds RAP as well as lactoferrin. Binding of both ligands was abolished by the excess of unlabeled ligands (data not shown).


Figure 5: Ligand blots of LDL receptors with RAP and lactoferrin. A, chicken embryo fibroblasts were cultured in medium supplemented with lipoprotein-deficient serum in the presence of 2 µg/ml mevinolin (lanes 1 and 3) or in medium supplemented with fetal bovine serum in the presence of 25-OH-cholesterol (4 µg/ml) (lanes 2 and 4). Cell pellets were solubilized with Triton X-100 as described under ``Experimental Procedures.'' Aliquots of 50 µg of protein/lane were subjected to electrophoresis under nonreducing conditions, transferred to nitrocellulose, and incubated with I-LDL (250 cpm/ng; 10^6 cpm/ml; lanes 1 and 2) or I-RAP-GST (1 10^3 cpm/ng, 5 10^6 cpm/ml; lanes 3 and 4). Autoradiography was performed for 36 h. Positions of molecular mass standards are indicated. B, purified bovine LDL receptor (lane 1, 0.1 µg; lanes 2 and 3, 0.5 µg) was subjected to electrophoresis under nonreducing conditions, transferred to nitrocellulose, and incubated with 10 µg/ml anti-bovine LDL receptor antibody (lane 1), I-RAP-GST (1 10^3 cpm/ng, 5 10^6 cpm/ml; lane 2), and I-lactoferrin (1 10^3 cpm/ng, 5 10^6 cpm/ml; lane 3). Bound IgG was visualized with protein A-HRP and the chemiluminescence system as described under ``Experimental Procedures.'' Exposure time was 1 min. Autoradiography for lanes 2 and 3 was 48 h. Positions of molecular mass standards are indicated.



Having shown that RAP and lactoferrin bind to chicken OVR and RAP binds to the mammalian and chicken LDL receptor, we measured the binding affinities of these ligands to the LDL receptor and compared them to those for OVR. Analysis was performed on the BIAcore system (Pharmacia Biosensor) and was facilitated through the availability of purified chicken OVR and purified bovine LDL receptor. As a control ligand, we used an unrelated single chain antibody fragment. Representative original sensograms are shown in Fig. 6(Panel A, RAP binding to OVR; Panel B, RAP binding to LDL receptor; Panel C, lactoferrin binding to OVR; Panel D, unrelated single chain antibody binding to LDL receptor). k was obtained as ratio of k to k and is given in M. Calculations were performed assuming first order kinetics. As demonstrated in Table 1, RAP exerts a high affinity toward OVR with an affinity constant of 3 10^8M. RAP binds to the LDL receptor with lower affinity (5 10^7M). The binding affinity of lactoferrin, however, was two orders of magnitude smaller for OVR in comparison to RAP and could not be determined for the LDL receptor, indicating an affinity constant of smaller than 10^5M.


Figure 6: Determination of affinity constants for RAP and lactoferrin for OVR and LDL receptor by surface plasmon resonance. Analysis was carried out on a BIAcore device (Pharmacia Biosensor). Purified OVR and bovine LDL receptor were coupled to different CM5 sensor chips as described under ``Experimental Procedures'' giving 1000 response units (RU) for OVR and 250 RU for the LDL receptor, respectively. Representative original sensograms for RAP binding to OVR (Panel A), for RAP binding to LDL receptor (Panel B), for lactoferrin binding to OVR (Panel C), and for binding of an unrelated single chain antibody to the LDL receptor (Panel D) are shown. These sensograms were obtained at the following ligand concentrations: RAP, 2 10M; lactoferrin, 4 10M; single chain antibody fragment, 2 10M.





Since RAP to date has been demonstrated in mammals only, we investigated whether this intracellular protein is also expressed in chicken. We used an anti-human RAP antibody which was immunopurified on recombinant RAP for Western blot experiments. As shown in Fig. 7, this antibody reacts with recombinant human RAP (lane 1) as well as with the fusion protein (58 kDa, lane 2) used for most of the experiments described in this report. Chicken follicle membrane extracts (lane 3) and liver membrane extracts (lane 4) showed the same band as present in a rat endosomal fraction (lane 5) used as a control(55) . This experiment clearly demonstrates for the first time the presence of RAP in a non-mammalian species.


Figure 7: Western blots of membrane extracts prepared from chicken tissues and rat liver endosomes. Recombinant RAP (0.5 µg, lane 1) and recombinant RAP-GST (0.5 µg, lane 2), Triton X-100 extracts of chicken follicle membranes (5 µg of protein, lane 3), of chicken liver membranes (15 µg of protein, lane 4), and rat endosomes (5 µg of protein, lane 5) were subjected to electrophoresis under nonreducing conditions, transferred to nitrocellulose, and incubated with 10 µg/ml anti-human RAP antibody as described under ``Experimental Procedures.'' Bound IgG was visualized with protein A-HRP and the chemiluminescence system described under ``Experimental Procedures.'' Exposure times were 1 min for lanes 1 and 2, 10 min for lane 3, and 5 min for lanes 4 and 5. Positions of molecular mass standards are indicated.




DISCUSSION

The physiology of the laying hen, and probably of all egg laying species, is well adapted to the special needs brought about by the massive deposition of yolk precursors into the growing oocytes. Since the major yolk precursors are lipoproteins, i.e. VLDL and VTG, the hen's reproductive effort is closely related to its lipoprotein metabolism. Key players in this complex system are receptors belonging to the family of LDL receptor-related proteins. As outlined in the Introduction, the hen expresses at least two related pairs of these receptors, each pair specific for somatic cells and oocytes, respectively.

Here we compared receptor interaction of RAP and lactoferrin among members of the chicken LDL receptor gene family, in particular with OVR which is expressed at high levels in the growing female germ cell. As clearly demonstrated by ligand-blotting experiments, both proteins bind to chicken OVR. In addition, RAP also binds to the chicken somatic LRP/alpha(2)MR and to the chicken LDL receptor. Furthermore, degradation studies with cells expressing OVR showed that a VLDL receptor-type protein can specifically mediate endocytosis and lysosomal degradation of RAP. Unfortunately, degradation experiments with lactoferrin were not conclusive since non-receptor-mediated binding of lactoferrin to cells in tissue culture is too high, possibly due to binding to cell-surface glycosaminoglycans. (^2)This observation is in agreement with results published by Ziere et al. (77) showing that, on Chinese hamster ovary cells, low affinity binding sites for lactoferrin outnumber the specific sites by a factor of 1 10^3. This low affinity binding precedes specific receptor binding and retards consecutive internalization.

Interestingly, there is one member of the chicken LDL receptor gene family which, under the conditions used, does not bind either of the two ligands, the oocyte-specific LRP. The reason for the lack of binding is not yet clear. Two possibilities are worthwhile considering. (i) Since purified protein is not available, crude membrane extracts had to be used in these experiments. If the affinity of RAP toward oocyte-specific LRP is significantly lower than to OVR, binding may not be evident in such experiments, in analogy to the LDL receptor (see below). (ii) The primary structure, especially the number and/or arrangements of the binding repeats in this protein, may be different from the other RAP-binding members, rendering it unable to interact with RAP. This would be in agreement with our recent finding that oocytic LRP, in contrast to somatic LRP/alpha(2)MR and OVR, also does not recognize alpha(2)M(20) .

When used in binding competition studies, both proteins were able to block binding of all known ligands to OVR. This is particularly interesting for lactoferrin. Lactoferrin was reported to inhibit LRP/alpha(2)MR-mediated cholesteryl esterification elicited by apoE-enriched VLDL in cultured cells, but not to have any effect on the degradation of I-alpha(2)-macroglobulin in fibroblasts(56) . This is consistent with earlier in vivo results demonstrating that lactoferrin inhibits clearance of chylomicron remnants, but not of alpha(2)-macroglobulin(52, 78) . Apparently, chylomicron remnants as well as apoE-enriched VLDL and alpha(2)-macroglobulin bind to different sites on LRP/alpha(2)MR, most likely residing on different clusters of binding repeats, and lactoferrin can selectively discriminate between them. On the other hand, OVR contains a single cluster of 8 binding repeats and binds apoE (79) as well as alpha(2)-macroglobulin (20) , and binding of both ligands is abolished by lactoferrin. These results, together with OVR's ability to bind RAP with high affinity and rhinoviruses of the minor group (^3)and tissue plasminogen activator/plasminogen activator inhibitor complexes (^4)make the single binding repeat cluster of OVR the most universal ligand binding domain of all of these related proteins. From an evolutionary point of view, OVR could be a primordial molecule originally designed to serve as multifunctional yolk precursor receptor crucial to reproduction, from which more specialized receptors were derived(79) . This could have been achieved by either restricting the ligand-binding specificity by reducing the number of binding repeats, as in the case of LDL receptors, or by distributing specificities over more than one such cluster, the situation found in LRP/alpha(2)MR and gp330.

The cross-competition studies presented here revealed another interesting detail. When RAP was used as inhibitor at 1000-fold molar excess, binding of all yolk precursor ligands was completely abolished. In contrast, unlabeled yolk precursors at the same molar excess were ineffective in competing for RAP binding. Nevertheless, the affinity of RAP for OVR is in the same range as the affinities of VLDL or VTG(17) . Thus, these findings make it unlikely that RAP binds to the same site as do the yolk precursors. In other words, ligand binding inhibition by RAP cannot be explained by direct competition for the same site on OVR. We therefore propose, similarly to the situation discussed for the LDL receptor (76) and for LRP/alpha(2)MR(40) , a model in which RAP exerts its action on OVR by binding to a site different from the yolk precursors, thereby changing the conformation of the receptor and rendering it incompetent for binding the other ligands. Using in vitro binding assays, it was found that LRP/alpha(2)MR binds 2 RAP molecules per receptor(80) . However, other experiments suggested that under saturating conditions 6-7 RAP molecules bind to 1 LRP/alpha(2)MR molecule(30) . Independent of the true stoichiometry of RAP and LRP/alpha(2)MR, it remains to be established whether RAP's inhibiting effect on all known ligands for LRP/alpha(2)MR could also be explained by a similar model as proposed for OVR.

Considering recent results that show that RAP indeed binds to the LDL receptor, although with an affinity too low for successful ligand blotting(76) , it was surprising to see clear binding of RAP in ligand blots to the chicken LDL receptor expressed on induced fibroblasts. To confirm this finding and to study this interaction further, we used purified bovine LDL receptor for ligand blot experiments. Indeed, under the conditions used in this experiment, the mammalian LDL receptor bound RAP and lactoferrin as well. Since ligand blot experiments do not give quantitative results in terms of ligand binding affinities, we chose to use the BIAcore system to quantitativly assess and compare binding kinetics of RAP and lactoferrin to OVR as well as to the bovine LDL receptor. RAP binds to the LDL receptor with an affinity constant of 5 10^7M, a value 10 times higher than that published by Medh et al.(76) . This discrepancy can possibly be explained by the following considerations. First, we used bovine LDL receptor, whereas the study mentioned was carried out on human fibroblasts. Second, only partially purified LDL receptor preparations had been used in these experiments, whereas data presented here were obtained with pure receptor. Third, LDL receptors had been immobilized using IgG-C7, a monoclonal antibody that binds to the first binding repeat of the LDL receptor(61) . Although the first binding repeat does not play an essential role in binding of apoB and apoE, IgG-C7 might well interfere with and reduce the receptor binding affinity of RAP.

Since RAP has not yet been shown to be expressed in non-mammalian systems, we asked whether the interaction of RAP with LDL receptor members in the chicken could be of physiological relevance. Indeed, here we could demonstrate for the first time that RAP is expressed in chicken tissues also expressing either OVR or LRP/alpha(2)MR. If the function of RAP is related to the function of members of the LDL receptor gene family, it will be interesting to determine whether the expression of RAP can be demonstrated in lower animals such as Caenorhabditis elegans, which possesses a gene for LRP/alpha(2)MR(67) . The situation with lactoferrin in the chicken is even less clear; our search for the expression of a lactoferrin-type protein has not revealed decisive data. In eggs such a gene product might serve as a primitive immune barrier.

Nevertheless, the oocyte which expresses large amounts of a RAP- and lactoferrin-binding member of the LDL receptor family is a system expected to refine the actual physiological functions of these small proteins.


FOOTNOTES

*
This work was supported by the Austrian Science Foundation Grants P9508-MOB (to J. N.), S07108-MED, and European Communities Grant PL-931088 (to W. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

In memoriam to Michael O. Kaderli.

§
Supported by the Danish Science Research Council. Present address: Institute of Medical Biochemistry, University of Aarhus, Denmark.

To whom correspondence should be addressed: Dept. of Molecular Genetics, Biocenter and University of Vienna, Dr. Bohrgasse 9/II, A-1030 Vienna, Austria. Tel. 43-1-79515-2111; Fax: 43-1-79515-2900; JNIMPF{at}mol.univie.ac.at.

^1
The abbreviations used are: OVR, oocyte very low density lipoprotein/vitellogenin receptor; LDL, low density lipoprotein; VLDL, very low density lipoprotein; VTG, vitellogenin; apo, apolipoprotein; alpha(2)M, alpha(2)-macroglobulin; alpha(2)M*, trypsin-treated alpha(2)M; alpha(2)MR, alpha(2)M receptor; LRP, LDL receptor-related protein; RAP, receptor-associated protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

^2
M. Httinger, personal communication.

^3
D. Blaas, unpublished observation.

^4
M. Haumer, personal communication.


ACKNOWLEDGEMENTS

We appreciate the excellent technical support by Romana Kukina, Harald Rumpler, and Robert Wandl.


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