©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Very Low Density Lipoprotein Receptor Binds and Mediates Endocytosis of Urokinase-type Plasminogen Activator-Type-1 Plasminogen Activator Inhibitor Complex (*)

(Received for publication, December 8, 1994; and in revised form, May 24, 1995)

Christian W. Heegaard (1) Anna Carina Wiborg Simonsen (1) Kazuhiro Oka (2) Lars Kj (1) Anni Christensen (1) Bente Madsen (1) Lars Ellgaard (3) Lawrence Chan (2) Peter A. Andreasen (1)(§)

From the  (1)Department of Molecular Biology, University of Aarhus, DK-8000C, Aarhus, Denmark, the (2)Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030 and the (3)Laboratory of Gene Expression, University of Aarhus, DK-8000 C Aarhus, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PAbulletPAI-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-PAbulletPAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PAbulletPAI-1, severalfold higher than non-transfected parental CHO cells. u-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-1.


INTRODUCTION

The urokinase (u-PA)(^1)-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-PAbulletPAI-1 complex (u-PAbulletPAI-1) can be accomplished by binding to the endocytosis receptors alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha(2)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-PAbulletPAI-1 to the endocytosis receptors (see review by Andreasen et al. (1994)). The endocytosis receptors alpha(2)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 alpha(2)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(r) 130,000 species (Daniel et al., 1983) and VLDL-R as an M(r) 100,000-130,000 species (Battey et al., 1994; Wiborg Simonsen et al., 1994), while both alpha(2)MR/LRP and gp330 have M(r) 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(r). alpha(2)MR/LRP consists of an M(r) 515,000 extracellular ligand-binding alpha-chain and an M(r) 85,000 membrane-spanning beta-chain, while gp330 consists of a single amino acid chain of M(r) 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(r) 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 alpha(2)MR/LRP and gp330, several other, structurally unrelated ligands. An M(r) 40,000 receptor-associated protein (RAP) binds strongly to alpha(2)MR/LRP and gp330, and inhibits the binding of all other currently known ligands, including u-PAbulletPAI-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-PAbulletPAI-1. Transfection of CHO cells with VLDL-R cDNA confers them with expression of M(r) 130,000 variant of VLDL-R and an efficient, RAP-sensitive endocytosis of u-PAR-bound u-PAbulletPAI-1. A cell line with endogenous expression of M(r) 105,000 VLDL-R variant and devoid of alpha(2)MR/LRP and gp330 was also found to show efficient RAP-sensitive endocytosis of u-PAR-bound u-PAbulletPAI-1.


EXPERIMENTAL PROCEDURES

Proteins and Antibodies

Human u-PA was purchased from Serono (Aubonne, Switzerland). The following proteins were prepared as described previously: diisopropyl fluorophosphate-inhibited u-PA (DFP-u-PA) (Jensen et al., 1990), human PAI-1 (Munch et al., 1991, 1993), u-PAbulletPAI-1 (Moestrup et al., 1993a; Nykjet al., 1994a), and human recombinant RAP (Nykjet al., 1992). Human alpha(2)-macroglobulin-methylamine (alpha(2)M*) (Sottrup-Jensen et al., 1980) and rat alpha(1)-inhibitor-3-chymotrypsin complex (alpha(1)I(3)bulletCT) (Sottrup-Jensen et al., 1989) were gifts from Dr. L. Sottrup-Jensen, University of Aarhus, Denmark. Human alpha(2)MR/LRP (Nykjet al., 1992) was a gift from Drs. A. Nykjand J. Gliemann, University of Aarhus, Aarhus, Denmark. Human PAI-2 (et al., 1986) was a gift from Dr. I. Lecander, University of Lund, Lund, Sweden. Recombinant human u-PAR was a gift from Dr. R. L. Cohen, Cancer Research Institute, University of California, San Francisco, CA (Nykjet al., 1994a).

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 10^6 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).

Purification of RAP-binding Proteins from Cell Membranes

Large scale preparation of cell membranes from bovine mammary gland and small scale preparation of cell membranes from cultured cells were performed as described earlier (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 NaH(2)PO(4), pH 7.4, 124 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl(2), 1.2 mM MgSO(4), 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(2), 0.6% CHAPS. Bound protein was eluted with 0.1 M CH(3)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.

Electrophoresis

SDS-PAGE was performed by standard methods in 4-16% gradient gels. The following M(r) markers were used: myosin (M(r) 200,000), Escherichia coli beta-galactosidase (M(r) 116,000), phosphorylase b (M(r) 97,400), bovine serum albumin (M(r) 66,200), and ovalbumin (M(r) 43,000).

Ligand Blotting and Immunoblotting Analyses

Proteins were resolved by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride filters. The filters were then incubated with 30 pMI-labeled ligands, and in some cases with non-radioactive ligands in various concentrations, in buffer A supplemented with 0.5% bovine serum albumin (Nykjet al., 1994a). After washing, bound ligands were visualized by autoradiography. Spectrophotometric scanning of autoradiographic films was performed with a Shimadzu CS-930 dual wavelength scanner. By comparing the relative intensities of alpha(2)MR/LRP alpha-chain and VLDL-R protein bands in Coomassie Blue-stained polyacrylamide gels with the relative intensities of Coomassie Blue-stained protein bands after transfer to polyvinylidene difluoride filters, we estimated that M(r) 515,000 alpha(2)MR/LRP alpha-chain is transferred around 50% as efficiently as M(r) 105,000 VLDL-R.

For quantitative analysis of steady state ligand binding to VLDL-R and alpha(2)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 alpha(2)MR/LRP alpha-chain in single gel lanes were cut out. Individual strips were incubated with 1 ml of 10 pMI-u-PAbulletPAI-1 and increasing concentrations of non-radioactive u-PAbulletPAI-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 Kvalues 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.

Binding Experiments with Sepharose 4B-coupled Antibody

Approximately 0.5 ml of Sepharose 4B, coupled with approximately 1 mg of monoclonal anti-PAI-1 IgG from hybridoma clone 2, was incubated at 4 °C for 16 h with 1 mg of u-PAbulletPAI-1 in buffer A with 0.5% bovine serum albumin. After washing, 1 µg of purified RAP-binding proteins from bovine mammary gland membranes was added to the Sepharose, in a total volume of 700 µl of the above-mentioned buffer, followed by incubation for 16 h at 4 °C. The mixture was then centrifuged at 1,000 g for 5 min and the supernatant collected. The Sepharose 4B was washed with 10 ml of the same buffer, and bound protein eluted with 10 ml of the buffer also used for eluting RAP-columns (see above). Parallel incubations with uncoupled Sepharose served as a control. Supernatant, wash, and eluate were concentrated on a Centricon YM-30 membrane and assayed for VLDL-R by immunoblotting analysis.

Cell Culture, Cell Ligand Binding, and Degradation Experiments

In order to create cells stably transfected with human VLDL-R cDNA, human VLDL-R cDNA clone hv58 (Oka et al., 1994b) was digested with NotI (blunt-ended by mung bean nuclease) and HindIII. This fragment was subcloned into the BglII (blunt-ended)/HindIII sites of the expression vector pCMV4 (Anderson et al., 1989). Transfection into CHO-ldlA7 cells (Kingley and Krieger, 1984), a mutant Chinese hamster ovary cell line lacking LDL-R (generously provided by Dr. M. Krieger, Massachusetts Institute of Technology), was carried out with 3.8 µg of pCMV4 containing human VLDL-R cDNA and 0.2 µg of pMC neo poly(A) (Stratagene) by using Lipofectin (Life Technologies, Inc.) according to the manufacturer's instructions. Stable transfectants were selected in medium supplemented with 0.4 mg/ml G418 (Life Technologies, Inc.).

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 10^4 and 10^6 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).

Analysis of Receptor-Ligand Binding Data

Dissociation constants K for ligand-receptor binding and the total receptor concentrations [R](0) were found by analyzing the concentration dependence of steady state binding in filter binding or whole cell binding experiments. The ratios between the concentrations of bound and free ligand (B/F) were determined with 5-10 pM radiolabeled ligand plus several concentrations of non-radioactive ligand. The (B/F) values were then plotted semilogarithmically versus the free ligand concentrations. The data were fitted by the method of least squares to the equation (B/F) = [RL]/[L] = [R](0)/(K + [L]), derived under the assumption of the simple binding equilibrium R + L right over left harpoon RL. R represents the receptor, L the ligand, and RL the receptor-ligand complex. This procedure yields the K and [R](0) values giving the best fit to the data.


RESULTS

Ligand Blotting Analysis of the Binding of u-PA-Inhibitor Complexes to VLDL-R from Bovine Mammary Gland

Fig. 1(lane2) shows SDS-PAGE and ligand blotting analysis of RAP binding to proteins purified from bovine mammary gland cell membranes by affinity chromatography with RAP immobilized on Sepharose. The preparation contained a RAP binding activity caused by the ligand binding M(r) 515,000 alpha-chain of alpha(2)MR/LRP, migrating near the top of the gel, and a RAP binding activity migrating in a position corresponding to M(r) 105,000. We recently showed that the latter binding activity represents VLDL-R, as judged by its N-terminal amino acid sequence and reactivity with a rabbit polyclonal peptide antibody against the C terminus of VLDL-R (Wiborg Simonsen et al., 1994). The relationship of this binding activity to VLDL-R was confirmed here by immunoblotting analysis with the anti-VLDL-R antibody (Fig. 1, lanes7 and 8).


Figure 1: Ligand blotting analysis of the binding of u-PA-inhibitor complexes to RAP-binding proteins from bovine mammary gland membranes. Purified alpha(2)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(r) 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-PAbulletPAI-1. One comigrated with alpha(2)MR/LRP alpha-chain. The capacity of u-PAbulletPAI-1 to bind to alpha(2)MR/LRP alpha-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(r) 105,000). There was no binding of radiolabeled DFP-u-PA or u-PAbulletPAI-2 complex (u-PAbulletPAI-2). The binding of radiolabeled u-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-1 and not a contaminant in the ligand preparation. The effects of the monoclonal antibodies on the binding to alpha(2)MR/LRP alpha-chain in the preparations were largely in agreement with previous observations (Nykjet al., 1994a). The effects on the binding to VLDL-R and alpha(2)MR/LRP alpha-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-PAbulletPAI-1 to VLDL-R but inhibited weakly its binding to alpha(2)MR/LRP a-chain.


Figure 2: Effect of monoclonal anti-PAI-1 antibodies and anti-u-PA antibodies on the binding of u-PAbulletPAI-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 pMI-u-PAbulletPAI-1, in the presence of 20 µg/ml of the indicated monoclonal antibodies. M(r) markers are indicated on the left.



The observed effect of monoclonal anti-PAI-1 from hybridoma clone 2 was utilized to confirm that u-PAbulletPAI-1 was actually binding to VLDL-R and not to another, comigrating RAP-binding protein (Fig. 3). In the presence of non-radioactive u-PAbulletPAI-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(r) 105,000 VLDL-R in the original preparation.


Figure 3: Binding of VLDL-R in complex with u-PAbulletPAI-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-PAbulletPAI-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(r) markers are indicated on the left.



Fig. 4shows ligand blotting analysis of the ability of various compounds to inhibit u-PAbulletPAI-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 alpha(2)MR/LRP alpha-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-PAbulletPAI-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-PAbulletPAI-1 and the indicated non-radioactive compounds. PAI-1 was in the latent form. M(r) markers are indicated on the left.



To compare the binding affinity of u-PAbulletPAI-1 for VLDL-R with the binding affinity for alpha(2)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 alpha(2)MR/LRP alpha-chain, respectively, were used for determining the concentration dependence of steady state binding of u-PAbulletPAI-1 to the two different receptors (Fig. 5). The u-PAbulletPAI-1 concentration giving half-saturation (the apparent K) of VLDL-R was approximately 1.5 nM and the corresponding value for binding to alpha(2)MR/LRP alpha-chain was approximately 0.5 nM. The later value is in good agreement with the values previously reported for binding of u-PAbulletPAI-1 to alpha(2)MR/LRP on cell surfaces (around 1 nM) or to alpha(2)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 alpha(2)MR/LRP alpha-chain.


Figure 5: Concentration dependence of u-PAbulletPAI-1 binding to VLDL-R and alpha(2)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 alpha(2)MR/LRP alpha-chain were cut out from single gel lanes. The individual strips were incubated with 1 ml of buffer with 10 pMI-u-PAbulletPAI-1 and increasing concentrations of the same ligand in non-radioactive form. The maximal amount of ligand bound was around 3% for binding to alpha(2)MR/LRP alpha-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](0)/(K + [L]). The K values corresponding to the fitted curves are indicated in the figure. For further details, see text.



Endocytosis of u-PAR-bound u-PAbulletPAI-1 in CHO Cells Stably Transfected with VLDL-R cDNA

Parental CHO cells and CHO cells stably transfected with a plasmid directing expression of human VLDL-R were characterized with respect to expression of endocytosis receptors and u-PAR by the use of ligand blot and whole cell binding assays with RAP, DFP-u-PA, and u-PAbulletPAI-1. RAP binds specifically to the endocytosis receptors (Nykjet al., 1992, and above). DFP-u-PA binds with a high affinity to u-PAR (Jensen et al., 1990), while the affinity of DFP-u-PA to alpha(2)MR/LRP and VLDL-R is very low, 100 nM, having at most a slight effect on binding of u-PAbulletPAI-1 (see Nykjet al. (1994a) and above). u-PAbulletPAI-1 binds to u-PAR with the same affinity as DFP-u-PA (Jensen et al., 1990) and was expected to partition between u-PAR and the endocytosis receptors according to the relative affinities to and the relative concentrations of each of receptor type, as predicted by the binding equation (see ``Experimental Procedures'').

Ligand blotting analysis showed that transfected CHO cells expressed an abundant RAP binding activity of M(r) 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(r) range 200,000-600,000, probably due to aggregated material (Fig. 6, A-C). RAP binding activity that could be ascribed to alpha(2)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(r) 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(r) 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 (bullet), at a cell density of 10^4 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](0)/(K + [L]). The K and [R](0) 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 (10^4 cells/well) were incubated with 10 pM radiolabeled u-PAbulletPAI-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(r) 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-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-1 to the two receptors and their relative amounts, as determined by the whole cell binding assays above.

The endocytic capacity for u-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-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 (10^4 cells/well) were incubated at 37 °C with 30 pMI-labeled u-PAbulletPAI-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(r) 130,000 VLDL-R-mediated endocytosis of u-PAR-bound u-PAbulletPAI-1, while there was no or little endocytosis of DFP-u-PA. Fluid phase u-PAbulletPAI-1 could also be taken up directly by VLDL-R.

Endocytosis of u-PAR-bound u-PAbulletPAI-1 in MCF-7 Cells

PMA-treated MCF-7 cells were used to investigate whether M(r) 105,000 VLDL-R, expressed endogenously by mammary epithelial cell lines (Wiborg Simonsen et al., 1994), was able to mediate cellular endocytosis of u-PAR-bound u-PAbulletPAI-1.

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(r) 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(r) 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 alpha(2)MR/LRP and gp330, because RAP-binding bands were absent in ligand blots at the positions of alpha(2)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 alpha(2)M rendered alpha(2)MR/LRP binding by methylamine treatment (alpha(2)M*) (Imber and Pizzo, 1981; Kaplan et al., 1981) or of rat alpha(1)I(3) rendered alpha(2)MR/LRP binding by chymotrypsin treatment (alpha(1)I(3)bulletCT) (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 alpha(2)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-PAbulletPAI-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-PAbulletPAI-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-PAbulletPAI-1 is endocytosed. This is in agreement with the much lower level of VLDL-R. No endocytosis (<0.1%) of alpha(2)M* and of alpha(1)I(3)bulletCT by MCF-7 cells could be measured (data not shown), again confirming the absence of alpha(2)MR/LRP in these cells. We conclude that M(r) 105,000 VLDL-R can mediate endocytosis of u-PAR-bound u-PAbulletPAI-1 and is incapable of mediating the endocytosis of alpha(2)M* and alpha(1)I(3)bulletCT.


Figure 10: Endocytosis of RAP and of u-PAbulletPAI-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.




DISCUSSION

The present report describes a novel endocytosis mechanism for u-PAR-bound u-PAbulletPAI-1, in addition to the previously described alpha(2)MR/LRP- and gp330-mediated ones (see review by Andreasen et al.(1994)). We show that purified VLDL-R binds u-PAbulletPAI-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-PAbulletPAI-1; we found that transfection of VLDL-R cDNA into CHO cells confers them with an efficient RAP-sensitive endocytosis of u-PAbulletPAI-1. Moreover, we have shown a RAP-sensitive endocytosis of u-PAR-bound u-PAbulletPAI-1 in MCF-7 cells, in which the only detectable RAP-binding receptor was VLDL-R. MCF-7 cells neither bound nor endocytosed the alpha(2)MR/LRP-ligands alpha(2)M* and alpha(1)I(3)bulletCT.

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 alpha(2)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 alpha(2)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(r) 105,000 and the M(r) 130,000 forms of VLDL-R, both of which bind and mediate the endocytosis of u-PAbulletPAI-1. Only the M(r) 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(r) 130,000 form.

Our present results show that the specificities of binding of components of the plasminogen activation system to VLDL-R and alpha(2)MR/LRP alpha-chain are similar. A noteworthy difference was observed with monoclonal anti-PAI-1 antibody from hybridoma clone 2, which markedly stimulated binding of u-PAbulletPAI-1 to VLDL-R but inhibited its binding alpha(2)MR/LRP alpha-chain. Moreover, the measurements of binding affinities for u-PAbulletPAI-1 revealed slightly lower affinity to VLDL-R than to alpha(2)MR/LRP alpha-chain. These observations suggest differences in the contact points of the ligands to the two receptors.

Importantly, the affinity of u-PAbulletPAI-1 for both alpha(2)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-PAbulletPAI-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.


FOOTNOTES

*
This work was supported by the Danish Cancer Society, the Danish Medical Research Council, and the Danish Biotechnology Programme (grants to P. A. A.) and by National Institutes of Health Grants HL16512 and HL27341 (to L. C.). 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.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, University of Aarhus, 130 C. F. M's Allé, DK-8000 C Aarhus, Denmark. Tel.: 45-89422680; Fax: 45-86196500.

(^1)
The abbreviations used are: u-PA, urokinase-type plasminogen activator; alpha(1)I(3), alpha(1)-inhibitor-3; alpha(1)I(3)bulletCT, alpha(1)I(3)-chymotrypsin complex; alpha(2)M, alpha(2)-macroglobulin; alpha(2)M*, alpha(2)M-methylamine; alpha(2)MR/LRP, alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein; CHAPS, 3-[(3-cholamido-propyl)dimethyl- ammonio]-1-propanesulfonate; DFP-u-PA, diisopropyl fluorophosphate-inhibited urokinase-type plasminogen activator; gp330, glycoprotein 330; LDL-R, low density lipoprotein receptor; PAI-1, type-1 plasminogen activator inhibitor; PAI-2, type-2 plasminogen activator inhibitor; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; RAP, M(r) 40,000 receptor-associated protein; PAGE, polyacrylamide gel electrophoresis; t-PA, tissue-type plasminogen activator; t-PAbulletPAI-1, t-PAbulletPAI-1 complex; u-PAbulletPAI-1, u-PAbulletPAI-1 complex; u-PAbulletPAI-2, u-PAbulletPAI-2 complex; u-PAR, urokinase-type plasminogen activator receptor; VLDL-R, very low density lipoprotein receptor.


ACKNOWLEDGEMENTS

We thank Dr. P. Armstrong for a critical reading of the manuscript and Drs. R. L. Cohen, J. Gliemann, I. Lecander, M. Z. Kounnas, M. Krieger, A. Nykj, L. Sottrup-Jensen, and D. K. Strickland for the gift of reagents.


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