(Received for publication, December 8, 1994; and in revised form, May 24, 1995)
From the
Very low density lipoprotein receptor (VLDL-R) was found to be
expressed in bovine mammary gland and the human breast carcinoma cell
line MCF-7 as an M 105,000 variant, and in Chinese
hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M
130,000 variant. The receptor was purified by
ligand affinity chromatography with immobilized M
40,000 receptor-associated protein (RAP). The purified receptor
was found to bind urokinase-type plasminogen activator-type-1
plasminogen activator inhibitor complex (u-PA
PAI-1), while there
was no or very weak binding of active site blocked u-PA (DFP-u-PA),
PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The
binding of u-PA
PAI-1 was blocked by RAP. The transfected CHO
cells had an efficient, RAP-sensitive endocytosis of u-PA
PAI-1,
severalfold higher than non-transfected parental CHO cells.
u-PA
PAI-1 endocytosis was partially inhibited by DFP-u-PA, which
blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA
sensitive u-PA
PAI-1 endocytosis was also observed in MCF-7 cells,
which were without detectable levels of other RAP-binding endocytosis
receptors. These results show that VLDL-R represents a novel
endocytosis mechanism for u-PA receptor-bound u-PA
PAI-1.
The urokinase (u-PA)()-mediated pathway of
plasminogen activation is implicated in extracellular proteolysis
during cell migration and invasion and extracellular matrix turn-over
(see review by Danø et al.(1985)). It is associated
with cell surfaces. Pro-u-PA, the zymogen form of u-PA, accumulates at
the cell surface by binding to the urokinase receptor (u-PAR). Binding
accelerates conversion of pro-u-PA to active u-PA and the u-PA-mediated
conversion of plasminogen to plasmin (see review by Fazioli and Blasi
(1994)). u-PA-mediated plasminogen activation is limited in time and
space by two inhibitors, PAI-1 and PAI-2. Both inhibitors are able to
react with u-PAR-bound u-PA, resulting in formation of u-PAR-bound
u-PA-inhibitor complexes (see review by Andreasen et al. (1990)).
u-PAR-bound pro-u-PA and active u-PA are cleared only
slowly from the cell surface. However, u-PAR-bound u-PA-inhibitor
complexes are rapidly endocytosed and degraded in many cell types.
Endocytosis of u-PAPAI-1 complex (u-PA
PAI-1) can be
accomplished by binding to the endocytosis receptors
-macroglobulin receptor/low density lipoprotein
receptor-related protein (
MR/LRP) and glycoprotein 330
(gp330). The glycosyl phosphatidylinositol-anchored u-PAR appears to be
unable to mediate endocytosis on its own but apparently transfers bound
u-PA
PAI-1 to the endocytosis receptors (see review by Andreasen et al. (1994)). The endocytosis receptors
MR/LRP and gp330 show low affinity to u-PAR-bound
pro-u-PA and u-PA, accounting for their resistance to endocytotic
clearance from the cell surface (Nykjet al., 1994a,
1994b).
Both MR/LRP and gp330 are members of the
low density lipoprotein receptor (LDL-R) family of endocytosis
receptors. The fourth mammalian member of the family is very low
density lipoprotein receptor (VLDL-R) (see review by Moestrup(1994)).
On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), LDL-R migrates as
an M
130,000 species (Daniel et al.,
1983) and VLDL-R as an M
100,000-130,000
species (Battey et al., 1994; Wiborg Simonsen et al.,
1994), while both
MR/LRP and gp330 have M
values of approximately 600,000 (Moestrup,
1994). All these receptors have a small, C-terminal cytoplasmic domain,
a trans-membrane domain, and a large, extracellular, ligand-binding
region. The different sizes of the extracellular portions account for
the differences in M
.
MR/LRP
consists of an M
515,000 extracellular
ligand-binding
-chain and an M
85,000
membrane-spanning
-chain, while gp330 consists of a single amino
acid chain of M
600,000 (see reviews by
Moestrup(1994) and Strickland et al.(1994)). The structures of
LDL-R and VLDL-R are very similar to each other, the main difference
being a slightly larger M
of the extracellular
portion of VLDL-R. In addition, VLDL-R mRNA has been reported to exist
in two splice variants, differing by the presence or absence of a
domain with potential O-linked glycosylation sites (Takahashi et al., 1992; Bujo et al., 1994; Oka et al.,
1994a; Sakai et al., 1994; Webb et al., 1994).
The
receptors of the LDL-R family function as endocytosis receptors for
various types of lipoproteins and, at least in the case of
MR/LRP and gp330, several other, structurally
unrelated ligands. An M
40,000 receptor-associated
protein (RAP) binds strongly to
MR/LRP and gp330, and
inhibits the binding of all other currently known ligands, including
u-PA
PAI-1 (see reviews by Andreasen et al.(1994),
Gliemann et al.(1994), Moestrup(1994), and Strickland et
al.(1994)). LDL-R has been reported to bind RAP with a low
affinity (Mokuno et al., 1994). VLDL-R was recently shown to
bind RAP with a high affinity and to mediate its endocytosis (Battey et al., 1994; Wiborg Simonsen et al., 1994).
We
show here that purified VLDL-R binds u-PAPAI-1. Transfection of
CHO cells with VLDL-R cDNA confers them with expression of M
130,000 variant of VLDL-R and an efficient,
RAP-sensitive endocytosis of u-PAR-bound u-PA
PAI-1. A cell line
with endogenous expression of M
105,000 VLDL-R
variant and devoid of
MR/LRP and gp330 was also found
to show efficient RAP-sensitive endocytosis of u-PAR-bound
u-PA
PAI-1.
Labeling of proteins with I and preparation of complexes between
I-labeled u-PA and the inhibitors were as described
(Jensen et al., 1990; Nykjet al., 1992,
1994a). The specific activities were approximately 2.5
10
Ci/mol.
Monoclonal mouse anti-PAI-1 antibodies from hybridoma clones 2, 3, and 5-7 and anti-u-PA antibodies from hybridoma clones 2, 6, and 12 were described previously (Nielsen et al., 1986; Keijer et al., 1990; Munch et al., 1991; Nykjet al., 1994a). A rabbit polyclonal antibody, prepared against a synthetic peptide ASVGHTYPAISVVSTDDDLA, which represents the C terminus of human and rabbit VLDL-R (Takahashi et al., 1992; Sakai et al., 1994), was a gift from Drs. M. Z. Kounnas and D. K. Strickland, American Red Cross, Washington, DC.
All other proteins and antibodies were those previously described (Jensen et al., 1990; Munch et al., 1991, 1993; Nykjet al., 1992, 1994a, 1994c; Heegaard et al., 1994; Wiborg Simonsen et al., 1994).
For purification of RAP-binding proteins,
cell membranes from 0.5 kg of bovine mammary gland tissue were
solubilized by homogenization in 1-2 liters of buffer A: 20
mM Hepes, 2.5 mM NaHPO
, pH
7.4, 124 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl
, 1.2 mM MgSO
, in this case
supplemented with 0.1% Triton X-100 and 0.5 mM
phenylmethylsulfonyl fluoride. The solution was centrifuged at 10,000
g and passed through a 1.2-µm filter to remove
insoluble material.
Cell membrane extract was applied to a 4-ml
column of Sepharose 4B-coupled with approximately 10 mg of RAP and
equilibrated in the buffer used for membrane solubilization. The column
was washed with 0.1 M Tris, pH 7.8, 0.14 M NaCl, 2
mM CaCl, 0.6% CHAPS. Bound protein was eluted with
0.1 M CH
COOH, pH 4.0, 0.5 M NaCl, 10
mM EDTA, 0.6% CHAPS. The eluate was neutralized with 0.1
volumes of 1.0 M Tris, pH 9.0. Approximately 0.5 mg of protein
was obtained from 0.5 kg of bovine mammary gland by eight consecutive
runs of the membrane extract over the column.
For
quantitative analysis of steady state ligand binding to VLDL-R and
MR/LRP, the purified RAP-binding proteins from bovine
mammary gland membranes were resolved by SDS-PAGE (65 ng of protein/gel
lane) and transferred electrophoretically to polyvinylidene difluoride
filters. Filter strips corresponding to VLDL-R and
MR/LRP
-chain in single gel lanes were cut out.
Individual strips were incubated with 1 ml of 10 pM
I-u-PA
PAI-1 and increasing concentrations of
non-radioactive u-PA
PAI-1 in buffer A supplemented with 0.5%
bovine serum albumin. The amounts of bound and unbound radioactive
ligands after the incubations were determined by
-counting. The
relative amounts of bound radioactive ligands were in some cases
determined by spectrophotometric scanning of autoradiographic films.
The bound-to-free ligand ratios (B/F) were used to
derive K
values as described below.
The same filter type was used for immunoblotting analysis, following a standard procedure with a primary polyclonal rabbit antibody, a secondary peroxidase-conjugated antibody, and visualization of bound antibody with the ECL immunodetection kit.
The following cell lines were from American Type Culture Collection: COS-1 (ATCC CRL 1650), HeLa (ATCC CCL 2), Hep2 (ATCC CCL 23), HepG2 (ATCC HB 8065), HT-1080 (ATCC CCL 121), JAR (ATCC HTB 144), LNCaP (ATCC CRL 1740), MCF-7 (ATCC HTB 22), PC3 (ATCC CRL 1435), and T47D (ATCC HTB 133).
The cells were cultured in Dulbecco's
modified Eagle's medium with 10% fetal calf serum, as described
previously (Knudsen et al., 1994; Kjet
al., 1995), except that the CHO cells were cultured in Ham's
F-12 medium with 5% fetal bovine serum. For binding and degradation
experiments, the cells were grown in 24-well dishes with 1.5-cm
diameter wells to a density of between 10 and 10
cells/well, the density depending on the purpose of the
experiment. When MCF-7 cells were used, an additional 32-h incubation
was performed under serum-free conditions with or without phorbol
12-myristate 13-acetate (PMA). For the experiments, the desired ligands
were added to the cells in 300 µl of buffer A with 0.5% bovine
serum albumin. For binding experiments, the cells were then incubated
for 16 h at 4 °C, at which time the media were collected, the cells
washed with ice-cold binding buffer and solubilized with 1 M NaOH, and the radioactivity in the media and bound to the cells
determined. For determination of cell-mediated degradation of ligands,
the cells were incubated at 37 °C for various time periods. Intact
and degraded ligand were then determined as the trichloroacetic
acid-precipitable and -soluble radioactivity, respectively. The amount
of degraded ligand was expressed as percentage of the total amount of
radioactivity added to the culture. For these experiments, cell
densities were chosen in a range in which binding and degradation were
proportional to cell number. All binding and degradation data for
radioactive ligands were corrected for the background obtained with a
3,000-10,000 excess of the same non-radioactive ligand (<5% of
the total amount of radioactive ligand).
Figure 1:
Ligand
blotting analysis of the binding of u-PA-inhibitor complexes to
RAP-binding proteins from bovine mammary gland membranes. Purified
MR/LRP (50 ng; lane1) and purified
RAP-binding proteins from bovine mammary gland membranes (65 ng/gel
lane; lanes 2-8) were resolved by SDS-PAGE in
4-16% gradient gels. Lanes1-6 show
ligand blotting analysis with the indicated radioactive and
non-radioactive ligands. Lanes7 and 8 show
immunoblotting analysis with rabbit polyclonal anti-VLDL-R IgG and
non-immune rabbit IgG, respectively. The exposure time for the ligand
blots with
I-RAP was approximately 2-fold shorter than
that for the other ligand blots. M
markers are
indicated on the left.
We have
now tested the ability of VLDL-R to bind u-PA and u-PA-inhibitor
complexes, using ligand blotting analysis with the purified RAP-binding
proteins from bovine mammary gland membranes. Fig. 1(lanes3-6) shows that the preparation contained two
binding activities for radiolabeled u-PAPAI-1. One comigrated
with
MR/LRP
-chain. The capacity of
u-PA
PAI-1 to bind to
MR/LRP
-chain is in
agreement with previous observations (Herz et al., 1992;
Nykjet al., 1992; Orth et al., 1992). The
other binding activity comigrated with VLDL-R (M
105,000). There was no binding of radiolabeled DFP-u-PA or
u-PA
PAI-2 complex (u-PA
PAI-2). The binding of radiolabeled
u-PA
PAI-1 could be blocked with an excess of non-radioactive RAP.
We tested the effect of monoclonal anti-PAI-1 and anti-u-PA
antibodies on u-PAPAI-1 binding (Fig. 2). Most of the
monoclonal antibodies either inhibited or stimulated the binding in the
position of VLDL-R. This confirmed that the radioactive molecule
binding in that position was indeed u-PA
PAI-1 and not a
contaminant in the ligand preparation. The effects of the monoclonal
antibodies on the binding to
MR/LRP
-chain in the
preparations were largely in agreement with previous observations
(Nykjet al., 1994a). The effects on the binding to
VLDL-R and
MR/LRP
-chain were similar but not
identical. The most striking difference was with anti-PAI-1 IgG from
hybridoma clone 2, which stimulated strongly the binding of
u-PA
PAI-1 to VLDL-R but inhibited weakly its binding to
MR/LRP a-chain.
Figure 2:
Effect of monoclonal anti-PAI-1 antibodies
and anti-u-PA antibodies on the binding of u-PAPAI-1 to
RAP-binding proteins from bovine mammary gland membranes. Purified
RAP-binding proteins from bovine mammary gland membranes (65 ng/gel
lane) were resolved by SDS-PAGE in 4-16% gradient gels. Ligand
blotting analysis was performed with 30 pM
I-u-PA
PAI-1, in the presence of 20 µg/ml of the
indicated monoclonal antibodies. M
markers are
indicated on the left.
The observed effect of monoclonal
anti-PAI-1 from hybridoma clone 2 was utilized to confirm that
u-PAPAI-1 was actually binding to VLDL-R and not to another,
comigrating RAP-binding protein (Fig. 3). In the presence of
non-radioactive u-PA
PAI-1, Sepharose coupled with that monoclonal
antibody bound VLDL-R from the preparation of RAP-binding proteins from
bovine mammary gland, as judged from the fact that immunoblotting
analysis of the bound fraction revealed a protein that reacted with the
anti-VLDL-R antibody and that comigrated with M
105,000 VLDL-R in the original preparation.
Figure 3:
Binding of VLDL-R in complex with
u-PAPAI-1 to monoclonal anti-PAI-1 IgG from hybridoma clone 2.
0.5 ml of Sepharose 4B, coupled with 1 mg of monoclonal anti-PAI-1
antibody from hybridoma clone 2 (lanes 1-3 and 1`-3`), and 0.5 ml plain Sepharose 4B (lanes4 and 4`) were incubated for 16 h at 4 °C
with 1 mg of non-radioactive u-PA
PAI-1, and then for 16 h at 4
°C with 1 µg of purified RAP-binding proteins from bovine
mammary gland membranes. Eluates from the Sepharose (lanes1 and 1` and lanes4 and 4`), the unbound fraction (lanes2 and 2`), and the wash (lanes3 and 3`)
were resolved by SDS-PAGE in 4-16% gradient gels and subjected to
immunoblotting analysis with anti-VLDL-R IgG (lanes 1-4)
or non-immune rabbit IgG (lanes 1`-4`). M
markers are indicated on the left.
Fig. 4shows ligand blotting analysis of the ability of
various compounds to inhibit u-PAPAI-1 binding to VLDL-R. The
binding was inhibited by EDTA, indicating Ca
dependence, and by the polyanion dextran sulfate. 100 nM
DFP-u-PA and latent PAI-1 were without measurable effect on the binding
to VLDL-R. 100 nM latent PAI-1 inhibited weakly (approximately
35%) the binding to
MR/LRP
-chain in the
preparation, in agreement with previous observations of a K
for binding of around 100 nM (Nykjet al., 1994a).
Figure 4:
Effect of various compounds on the binding
of u-PAPAI-1 to RAP-binding proteins from bovine mammary gland
membranes. Purified RAP-binding proteins from bovine mammary gland
membranes (65 ng of protein/gel lane) were resolved by SDS-PAGE and
subjected to ligand blotting analysis with
I-u-PA
PAI-1 and the indicated non-radioactive
compounds. PAI-1 was in the latent form. M
markers
are indicated on the left.
To compare the binding
affinity of u-PAPAI-1 for VLDL-R with the binding affinity for
MR/LRP, the RAP-binding proteins from bovine mammary
gland membranes were separated by SDS-PAGE and blotted onto filters.
Filter strips containing VLDL-R and
MR/LRP
-chain, respectively, were used for determining the concentration
dependence of steady state binding of u-PA
PAI-1 to the two
different receptors (Fig. 5). The u-PA
PAI-1 concentration
giving half-saturation (the apparent K
)
of VLDL-R was approximately 1.5 nM and the corresponding value
for binding to
MR/LRP
-chain was approximately
0.5 nM. The later value is in good agreement with the values
previously reported for binding of u-PA
PAI-1 to
MR/LRP on cell surfaces (around 1 nM) or to
MR/LRP immobilized in microtiter wells (around 0.4
nM) (Nykjet al., 1994a), showing that the
method employed here gives reliable results. These data suggest a
slightly lower affinity of the ligand to VLDL-R than to
MR/LRP
-chain.
Figure 5:
Concentration dependence of
u-PAPAI-1 binding to VLDL-R and
MR/LRP. Purified
RAP-binding proteins from bovine mammary gland membranes (65 ng/gel
lane) were separated by SDS-PAGE and transferred to filters. Filter
strips corresponding to VLDL-R and
MR/LRP
-chain
were cut out from single gel lanes. The individual strips were
incubated with 1 ml of buffer with 10 pM
I-u-PA
PAI-1 and increasing concentrations of the
same ligand in non-radioactive form. The maximal amount of ligand bound
was around 3% for binding to
MR/LRP
-chain and
1-2% for binding to VLDL-R. The bound to free ligand ratios (B/F), expressed as a fraction of the ratio found
with the lowest ligand concentration, were plotted semilogarithmically versus the free ligand concentrations. Each data point (mean
and standard deviation) corresponds to at least six determinations in
at least three independent experiments. The lines drawn were obtained
by fitting the data by the method of least squares to the equation B/F = [RL]/[L]
= [R]
/(K
+ [L]). The K
values corresponding to the fitted curves are indicated in
the figure. For further details, see text.
Ligand blotting analysis
showed that transfected CHO cells expressed an abundant RAP binding
activity of M 130,000, comigrating with VLDL-R
immunoreactivity. The RAP binding activity and the VLDL-R
immunoreactivity in that position in the parental, non-transfected
cells were at least 10-fold weaker. The blots also showed that the
transfected cells had some VLDL-R immunoreactivity and RAP binding
activity in the M
range 200,000-600,000,
probably due to aggregated material (Fig. 6, A-C). RAP binding activity that could be
ascribed to
MR/LRP was at least 5-fold lower that the
RAP binding activity caused by VLDL-R. Whole cell binding analysis
showed that the parental cells and the transfected cells had
approximately 30,000 and approximately 300,000 RAP binding sites/cell,
respectively, the K
values in both cases
being around 50 pM (Fig. 7A). These results,
taken together, show a strongly increased RAP binding activity due to M
130,000 VLDL-R expression in the transfected
cells.
Figure 6:
Ligand blotting and immunoblotting
analysis of VLDL-R in control CHO cells and CHO cells transfected with
VLDL-R cDNA. Membrane samples from wild type CHO cells (wt)
and CHO cells transfected with VLDL-R cDNA (tr) corresponding
to 10 µg of protein, 65 ng of purified RAP-binding proteins from
bovine mammary gland membranes (m), and 10 ng of purified
u-PAR (u) were resolved by SDS-PAGE in 4-16% gradient
gels and analyzed by immunoblotting and ligand blotting as indicated. M markers are indicated on the left.
Figure 7:
Whole cell binding assays of VLDL-R and
u-PAR in control CHO cells and CHO cells transfected with VLDL-R cDNA. A, wild type CHO cells () or CHO cells transfected with
VLDL-R cDNA (
), at a cell density of 10
cells/well,
were incubated for 16 h at 4 °C with 5 pM radiolabeled RAP
and varying concentrations of the same ligand in non-radioactive form.
The figure shows the ratios between the amounts of cell-bound and free
ligand (B/F) plotted semilogarithmically versus the
free ligand concentration. Means and standard deviations of triple
determinations are indicated. The lines drawn were obtained by fitting
the data to the equation B/F =
[R]/[L] =
[R]
/(K
+ [L]). The K
and [R]
values were: wild type cells,
43 pM and 30,600 receptors/cell; transfected cells, 42 pM and 295,700 receptors/cell. B, wild type and transfected
cells (10
cells/well) were incubated with 10 pM radiolabeled u-PA
PAI-1 and 100 nM of the indicated
non-radioactive compounds for 16 h at 4 °C, after which time the
ratios between the amounts of cell-bound and free ligand were
determined. Openbars indicate wild type cells; closedbars indicate transfected cells. Means and
standard deviations for four determinations in two independent
experiments are indicated.
Ligand blot analysis with radioactive DFP-u-PA showed that
the levels of u-PAR, migrating with M 50,000, were
indistinguishable in the two cell lines (Fig. 6D).
Whole cell binding assays with DFP-u-PA showed around 50,000 receptor
sites/cell and a K
value of 200 pM in both cell lines (data not shown).
Whole cell binding of 10
pM radioactive u-PAPAI-1 was approximately 2-fold higher
with transfected cells than with parental cells, and the additional
binding in the tranfected cells was inhibited by 100 nM non-radioactive RAP (Fig. 7B). In both cases, most
of the binding of this ligand could be almost totally inhibited by 100
nM non-radioactive DFP-u-PA plus 100 nM non-radioactive RAP. These observations demonstrate that the
transfected cells, but not the parental cells, bind a fraction of the
bound u-PA
PAI-1 directly to VLDL-R. Nearly all of the binding in
the parental cells was to u-PAR. The relative fractions bound to u-PAR
and VLDL-R were in good agreement with the prediction from the binding
equation, the relative affinities of u-PA
PAI-1 to the two
receptors and their relative amounts, as determined by the whole cell
binding assays above.
The endocytic capacity for u-PAPAI-1 in
the two cell lines was estimated from the conversion of
I-labeled ligands to trichloroacetic acid-soluble
radioactivity during incubation at 37 °C. The transfected CHO cells
degraded 30 pM radioactive u-PA
PAI-1 efficiently. The
degradation could be inhibited almost totally by 100 nM RAP
and partially by 100 nM DFP- u-PA. The DFP-u-PA and
RAP-inhibitable degradation of radioactive u-PA
PAI-1 by
transfected cells was more than 6-fold higher than in the
non-transfected cells (Fig. 8). Both cell lines showed little if
any degradation of DFP-u-PA (data not shown).
Figure 8:
Endocytosis of u-PAPAI-1 by control
CHO cells and CHO cells transfected with VLDL-R cDNA. Wild type CHO
cells and CHO cells transfected with VLDL-R cDNA (10
cells/well) were incubated at 37 °C with 30 pM
I-labeled u-PA
PAI-1, in the presence or
absence of the indicated non-radioactive ligands. After 4 h, the
percentages of degraded ligand in the media were determined. Openbars indicate wild type cells; closedbars indicate transfected cells. Means and standard deviations for
triple determinations are indicated.
Taken together, these
results demonstrate M 130,000 VLDL-R-mediated
endocytosis of u-PAR-bound u-PA
PAI-1, while there was no or
little endocytosis of DFP-u-PA. Fluid phase u-PA
PAI-1 could also
be taken up directly by VLDL-R.
As measured with ligand blotting analysis with DFP-u-PA, MCF-7 cells expressed very little u-PAR under basal conditions, but the level increased strongly upon PMA treatment (Fig. 9A). PMA-treated MCF-7 cells were found to contain approximately 90,000 u-PAR molecules/cell, using a whole cell binding assay with DFP-u-PA (data not shown).
Figure 9:
u-PAR and VLDL-R in MCF-7 cells. A, MCF-7 cell membranes (5 µg of protein/gel lane) from
control cells (lanes1 and 3) and cells
incubated with PMA for 32 h (lanes2 and 4)
were resolved by SDS-PAGE and subjected to ligand blotting analysis
with I-DFP-u-PA (lanes1 and 2) and
I-RAP (lanes3 and 4). M
markers are indicated on the left. B, whole cell assay of binding of
various ligands to MCF-7 cells. Confluent MCF-7 cells were incubated
for 16 h at 4 °C with the indicated radioactive and non-radioactive
ligands, after which time the concentrations of cell-bound ligand, as
percentages of the free ligand concentrations, were measured. Means and
standard deviations for at least eight determinations in at least two
independent experiments are indicated.
Control and
PMA-treated MCF-7 cells express M 105,000 VLDL-R
at the same level, as judged by RAP ligand blotting analysis. Both
control and PMA-treated MCF-7 cells appear to lack
MR/LRP and gp330, because RAP-binding bands were
absent in ligand blots at the positions of
MR/LRP and
gp330 (Fig. 9A; see also Wiborg Simonsen et al. (1994)). In whole cell binding assays, there was no measurable
saturable binding (<0.25%) to PMA-treated MCF-7 cells of human
M rendered
MR/LRP binding by
methylamine treatment (
M*) (Imber and Pizzo, 1981;
Kaplan et al., 1981) or of rat
I
rendered
MR/LRP binding by chymotrypsin
treatment (
I
CT) (Moestrup and
Gliemann, 1991) (Fig. 9B). These ligands did bind to
COS-1 cells (Herz et al., 1992; Orth et al., 1992;
Nykjet al., 1994a). These results confirmed the
absence of
MR/LRP in PMA-treated MCF-7 cells. Control
and PMA-treated MCF-7 cells contain approximately 2000 RAP-binding
sites/cell, as measured by whole cell binding assays (data not shown).
MCF-7 cells showed the highest VLDL-R expression among a number of cell
lines (COS-1, HeLa, HepG2, Hep2, HT-1080, JAR, LNCaP, PC3, and T47D)
analyzed by RAP ligand blotting analysis.
In whole cell binding
assays with PMA-treated MCF-7 cells, the binding of 10 pMI-u-PA
PAI-1 was almost completely inhibited by
100 nM DFP-u-PA, while no inhibition was observed with 100
nM RAP (data not shown), indicating that this ligand bound
exclusively to u-PAR, without any binding directly to VLDL-R. This is
in agreement with expectancies, considering the lower level of VLDL-R
in these cells, and in agreement with the results on non-transfected
CHO cells.
Fig. 10shows measurements of endocytosis of RAP,
u-PAPAI-1, and DFP-u-PA in PMA-treated MCF-7 cells. The results
obtained are very similar to those shown above for CHO-cells. The
endocytosis rates are lower in MCF-7 cells, and only u-PAR-bound
u-PA
PAI-1 is endocytosed. This is in agreement with the much
lower level of VLDL-R. No endocytosis (<0.1%) of
M*
and of
I
CT by MCF-7 cells could be
measured (data not shown), again confirming the absence of
MR/LRP in these cells. We conclude that M
105,000 VLDL-R can mediate endocytosis of
u-PAR-bound u-PA
PAI-1 and is incapable of mediating the
endocytosis of
M* and
I
CT.
Figure 10:
Endocytosis of RAP and of u-PAPAI-1
by MCF-7 cells. Serum-free cultures of confluent MCF-7 cells were
incubated at 37 °C with the indicated
I-labeled
ligands, in the presence or absence of the indicated non-radioactive
ligands. After 4 h, the percentages of degraded ligand in the media
were determined. Means and standard deviations for at least eight
determinations in at least two independent experiments are
indicated.
The present report describes a novel endocytosis mechanism
for u-PAR-bound u-PAPAI-1, in addition to the previously
described
MR/LRP- and gp330-mediated ones (see review
by Andreasen et al.(1994)). We show that purified VLDL-R binds
u-PA
PAI-1. The binding was observed both after resolution of
membrane proteins by SDS-PAGE and transfer to filters (ligand blotting
analysis) and by capture of the receptor by Sepharose coupled with a
monoclonal antibody against the ligand. In addition, we present
evidence that VLDL-R is able to mediate endocytosis of u-PAR-bound and
fluid-phase u-PA
PAI-1; we found that transfection of VLDL-R cDNA
into CHO cells confers them with an efficient RAP-sensitive endocytosis
of u-PA
PAI-1. Moreover, we have shown a RAP-sensitive endocytosis
of u-PAR-bound u-PA
PAI-1 in MCF-7 cells, in which the only
detectable RAP-binding receptor was VLDL-R. MCF-7 cells neither bound
nor endocytosed the
MR/LRP-ligands
M*
and
I
CT.
VLDL-R has a structure
very similar to that of LDL-R. The overall amino acid identity between
the two receptors is around 40%. In the extracellular portion, both
receptors contain, beginning at the N terminus, a cluster of complement
type repeats, an epidermal growth factor precursor homology domain, a
domain with O-linked sugars, a transmembrane domain, and an
intracellular domain. The ligand binding activity of LDL-R has been
shown to reside in the cluster of complement type repeats (see
Moestrup(1994) and Strickland et al.(1994)). The main
difference between LDL-R and VLDL-R is the eight complement type
repeats of VLDL-R, one more than LDL-R (Takahashi et al.,
1992; Bujo et al., 1994; Oka et al., 1994a; Sakai et al., 1994; Webb et al., 1994). This difference may
be important in conferring the two receptors with different ligand
specificities. A cluster of eight complement type repeats, similar to
the cluster in VLDL-R, is found near the N terminus of both
MR/LRP and gp330 (Moestrup, 1994; Saito et
al., 1994) and has been shown to be indispensable for binding RAP
and plasminogen activator-inhibitor complexes in
MR/LRP (Moestrup et al., 1993b; Willnow et al., 1994).
VLDL-R mRNA has been reported to exist in
two splice variants, which differ by the presence or absence of 84
nucleotides coding for the domain with potential O-linked
glycosylation sites (Sakai et al., 1994). We believe these two
mRNA are responsible for the M 105,000 and the M
130,000 forms of VLDL-R, both of which bind and
mediate the endocytosis of u-PA
PAI-1. Only the M
105,000 form has been detected in mammary gland and mammary
epithelial cell lines (Wiborg Simonsen et al., 1994), while
other cell lines and tissues, including the human Bowes cell lines and
bovine heart and brain (data not shown) express both forms. The
transfected CHO cells express predominantly the M
130,000 form.
Our present results show that the specificities
of binding of components of the plasminogen activation system to VLDL-R
and MR/LRP
-chain are similar. A noteworthy
difference was observed with monoclonal anti-PAI-1 antibody from
hybridoma clone 2, which markedly stimulated binding of u-PA
PAI-1
to VLDL-R but inhibited its binding
MR/LRP
-chain. Moreover, the measurements of binding affinities for
u-PA
PAI-1 revealed slightly lower affinity to VLDL-R than to
MR/LRP
-chain. These observations suggest
differences in the contact points of the ligands to the two receptors.
Importantly, the affinity of u-PAPAI-1 for both
MR/LRP and VLDL-R is much higher than that of the
non-complexed components. This explains the efficient endocytosis of
the complex and the absence of endocytosis of uncomplexed PAI-1 or u-PA
(Nykjet al., 1994a, 1994b). On this basis, PAI-1
inhibition of u-PAR-bound u-PA and endocytosis of u-PAR-bound
u-PA
PAI-1 may function to ensure a dynamic state of the u-PA
system at the cell surface, allowing regional and temporal variations
in the activity of the system. This may be necessary for optimal
operation of the system during cell migration and invasion.