(Received for publication, March 9, 1995)
From the
Receptor-associated protein (RAP) was originally described as a
39-kDa intracellular protein copurifying with mammalian low density
lipoprotein (LDL) receptor-related
protein/
We have characterized at the molecular and biochemical level the
chicken oocyte receptor (OVR) ( 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 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 A 39-kDa protein (receptor-associated protein,
RAP) was described which copurifies with LRP/ LRP/ 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
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
LDL receptor was purified from bovine
adrenal cortex by DEAE-cellulose and affinity chromatography on
LDL-Sepharose as described elsewhere (71) .
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),
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);
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/ 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 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
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
The finding that RAP, which was originally
described as ligand for LRP/
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
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)
Figure 4:
Cross-competition of RAP and lactoferrin
with VLDL, VTG, and
We then used labeled yolk
precursors and evaluated the cross-competition with RAP and
lactoferrin, respectively. As expected, As recently demonstrated, OVR also binds native and trypsin-treated
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/
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
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
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
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.
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/ 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/ 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/ 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/ 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 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/ 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.
In memoriam to
Michael O. Kaderli.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-macroglobulin receptor
(LRP/
MR). RAP has a high affinity for
LRP/
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/
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/
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
-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/
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.
)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
-macroglobulin
(
M)(20) .
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).
M-proteinase complexes (21, 22) (this receptor was therefore named LDL
receptor-related protein/
M receptor;
LRP/
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) .
MR from
liver and placenta (34, 35, 36) . RAP is an
intracellular protein with high affinity for LRP/
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/
MR
activity in vivo by associating with LRP/
MR
in recycling vesicles before the receptor reaches the plasma membrane.
RAP was successfully used in vivo to demonstrate
LRP/
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) .
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
-migrating very low
density lipoprotein(52, 53) . Interestingly, uptake of
activated
M was not blocked by lactoferrin in this
system. Using lactoferrin in ligand blots with rat liver and kidney
extracts, its binding to LRP/
MR and gp330 (megalin),
another member of the LDL receptor family(54) , was directly
demonstrated(55, 56) . Degradation of
I-
M in LDL receptor-negative fibroblasts
was strongly inhibited by RAP but not by lactoferrin(56) .
M. This, for lactoferrin, is in sharp contrast to its
interaction with LRP/
MR.
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 17-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/MR was obtained
using a synthetic peptide corresponding to the 14 carboxyl-terminal
amino acids of the heavy chain of chicken LRP/
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) .
M was purified
from citrated plasma of laying hens essentially according to
Sottrup-Jensen et al.(65) with minor modifications as
described previously(20) .
M-trypsin complexes
(
M*) were prepared by incubating native
M with a 15-fold molar excess of trypsin for 1 min
followed by addition of the same amount of soybean trypsin inhibitor.
Native
M was radioiodinated using the lactoperoxidase
method (66) to a specific activity of 900-1100 cpm/ng. Labeled
M-trypsin complexes were produced by reacting labeled
native
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
DH5
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 Na
I 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) .-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, 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
, 30 mM
CHAPS). Bound protein was eluted with a linear gradient (13.5 ml) of
buffer A containing 0-1 M NH
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
, 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.
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, 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
10
M and 2
10
M. Lactoferrin was used at
concentrations between 3
10
M and 1
10
M. 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) .
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/
MR (58) as described under ``Experimental Procedures.''
In lane 6, this antibody visualizes the somatic
LRP/
MR-515 in liver membranes, which comigrates with
the RAP-binding protein in lanes 4 and 5.
I-RAP-GST (0.5 µg/ml; specific
activity, 1
10
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
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
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.
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.
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) .
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°.
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.
MR, binds to OVR prompted
us to investigate whether lactoferrin, another ligand of
LRP/
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).
I-lactoferrin (1 µg/ml, 1
10
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.
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.
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
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,
M (0.5 mg/ml); lane 6,
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-
M (7 ng/ml, 13
10
cpm/ng) for Panel D and with
I-
M* (7 ng/ml, 12
10
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.
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.
M(20) . To further characterize this binding,
we performed competition experiments with RAP and lactoferrin. As
demonstrated in Fig. 4D for
I-
M and in Fig. 4E for
I-
M*, addition of RAP-GST competes for
the binding of both ligands (lanes 2). Lactoferrin also
interferes with the binding of
I-
M and
I-
M* (Fig. 4, D and E, lane 3), but to a somewhat smaller extent than
RAP under these conditions.
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
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).
I-LDL (250 cpm/ng; 10
cpm/ml; lanes 1 and 2) or
I-RAP-GST (1
10
cpm/ng, 5
10
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
cpm/ng, 5
10
cpm/ml; lane 2), and
I-lactoferrin (1
10
cpm/ng, 5
10
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.
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
M
. RAP binds to the LDL
receptor with lower affinity (5
10
M
). 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
M
.
10
M; lactoferrin, 4
10
M; single chain antibody fragment, 2
10
M.
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. (
)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
.
This low affinity binding precedes specific receptor binding and
retards consecutive internalization.
MR and OVR, also does not
recognize
M(20) .
MR-mediated
cholesteryl esterification elicited by apoE-enriched VLDL in cultured
cells, but not to have any effect on the degradation of
I-
-macroglobulin in
fibroblasts(56) . This is consistent with earlier in vivo results demonstrating that lactoferrin inhibits clearance of
chylomicron remnants, but not of
-macroglobulin(52, 78) . Apparently,
chylomicron remnants as well as apoE-enriched VLDL and
-macroglobulin bind to different sites on
LRP/
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
-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 (
)and tissue plasminogen
activator/plasminogen activator inhibitor complexes (
)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/
MR and gp330.
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/
MR binds 2 RAP molecules per
receptor(80) . However, other experiments suggested that under
saturating conditions 6-7 RAP molecules bind to 1
LRP/
MR molecule(30) . Independent of the true
stoichiometry of RAP and LRP/
MR, it remains to be
established whether RAP's inhibiting effect on all known ligands
for LRP/
MR could also be explained by a similar model
as proposed for OVR.
10
M
, 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.
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/
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.
M,
-macroglobulin;
M*, trypsin-treated
M;
MR,
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.
We appreciate the excellent technical support by
Romana Kukina, Harald Rumpler, and Robert Wandl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.