Regulation of the Ligand Binding Activity of the Human Very Low Density Lipoprotein Receptor by Protein Kinase C-dependent Phosphorylation*

Ramasamy SakthivelDagger §, Jing-Chuan ZhangDagger , Dudley K. Strickland||, Mats Gåfvels**, and Keith R. McCraeDagger DaggerDagger

From the Dagger  Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, || Holland Labs, American Red Cross, Rockville, Maryland 20855, and ** Center for Nutrition and Toxicology, Karolinska Institute at Huddinge University Hospital, S-14186 Huddinge, Sweden

Received for publication, May 9, 2000, and in revised form, August 23, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The very low density lipoprotein receptor (VLDL-R)1 is a member of the low density lipoprotein receptor (LDL-R) family, which encompasses several structurally related proteins such as the LDL receptor-related protein/alpha 2-macroglobulin receptor, gp330 (megalin), and apoE receptor 2 (1-4) among others. Members of this family are characterized by a cytoplasmic domain NPXY sequence that mediates ligand internalization through clathrin-coated pits (5) and an extracellular domain comprised of cysteine-rich complement-type and epidermal growth factor precursor-like repeats that regulate ligand binding specificity (2). Except for the presence of an additional complement-type repeat in its extracellular domain, the domain structure of the VLDL-R is identical to that of the LDL-R (6-8). Despite this homology, however, the ligand binding specificity of these receptors differs. Though both receptors bind apoE-containing lipoproteins (6, 7, 9, 10), only the LDL receptor binds lipoproteins containing apoB-100 (6, 7), whereas the VLDL-R binds several additional ligands such as lipoprotein lipase (9), (11) urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 (u-PA:PAI-1) complexes (11, 12), and Lp(a) (13). Finally, the 39-kDa receptor-associated protein (RAP), which co-purifies with (14, 15) and binds with high affinity to the LDL receptor-related protein/alpha 2-macroglobulin receptor (16-18), binds tightly to the VLDL-R (19) but with only low affinity to the LDL-R (19, 20).

Differences in the tissue distribution of the LDL and VLDL receptors also suggest non-overlapping physiologic functions. Although the LDL-R is highly expressed in liver and promotes LDL uptake and clearance (21), the VLDL-R is most abundant in heart and skeletal muscle (22), suggesting involvement in the uptake of triglyceride-rich lipoproteins in tissues dependent on fatty acid metabolism (2, 7, 8). However, the broad ligand specificity of the VLDL-R as well as the lack of coordinate regulation between the VLDL-R and lipoprotein lipase suggests that this receptor may also function in other processes distinct from lipid metabolism (23). In this context, the VLDL-R regulates cell surface urokinase receptor expression and urokinase receptor-dependent cellular migration (24).

We previously reported that the VLDL-R is expressed by human arterial and venous endothelial and smooth muscle cells in vitro and in vivo (11, 25) as well as by macrophage-derived foam cells within atherosclerotic plaques (25). Nakazato et al. (26) detected VLDL-R mRNA in smooth muscle cells and macrophages within atherosclerotic aorta from cholesterol-fed NZW and Watanabe hereditary hyperlipidemic rabbits (26). The VLDL-R is also present in the endothelium of capillaries, small arterioles, and coronary arteries of mouse and bovine origin (27). These studies suggest a potential role for the VLDL receptor in (patho)physiological vascular processes such as atherosclerosis.

Unlike that of the LDL-R, expression of the VLDL-R is not diminished by exogenous cholesterol (21, 28, 29). In choriocarcinoma cells, VLDL-R mRNA levels are regulated by insulin, 8-bromo-cAMP, and clofibrate (30), whereas in HL-60 cells, 1,25-dihydroxy vitamin D3 stimulates receptor expression (31). In vivo, thyroid hormone (32) and granulocyte-macrophage colony stimulating factor (33) stimulate the expression of VLDL-R mRNA in skeletal muscle, whereas estradiol stimulates VLDL-R expression in myocardium (34). However, changes in mRNA levels may not reflect the expression of a functional VLDL-R protein and whether the expression or activity of the VLDL-R may be regulated through post-transcriptional mechanisms has not been assessed. Here we report that the ligand binding affinity of the VLDL-R is diminished after protein kinase C (PK-C)-dependent phosphorylation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Tissue culture medium and reagents were from Mediatech (Herndon, VA). Fetal bovine serum was from Hyclone (Logan, Utah). The 39-kDa RAP, which co-purifies with the LDL receptor-related protein/alpha 2-macroglobulin receptor (14, 15) and inhibits ligand binding to this (16-18) as well as to the VLDL-R (19), was expressed and purified as described (17). For selected studies, RAP was conjugated to cyanogen bromide-activated Sepharose 4B (8.9 mg/ml of packed beads) per the manufacturer's protocol (Amersham Pharmacia Biotech) or labeled with 125I to a specific activity of ~5.0 × 106 cpm/µg using iodobeads. Affinity-purified anti-VLDL receptor antibodies, raised against a peptide corresponding to the C-terminal 20 amino acids of the human (35-37) mouse (38) and rabbit (6) VLDL-R, have been described (30). A rabbit antibody raised against a polypeptide encompassing the 160 N-terminal amino acids of the VLDL-R (25) was used for detection of a truncated VLDL-R mutant lacking the cytoplasmic domain. Anti-phosphoserine (1C8) and anti-phosphothreonine (1E11) monoclonal antibodies were from Biomol (Plymouth Meeting, PA), anti-phosphotyrosine antibodies were from Upstate Biotechnology (Lake Placid, NY), and anti-PK-C beta II antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Two-chain u-PA, PAI-1, and anti-PAI-1 antibodies were from American Diagnostica, Inc. (Greenwich, CT). Phorbol 12-myristate 13-acetate (PMA), protamine sulfate, streptavidin-peroxidase, and peroxidase-conjugated secondary antibodies were from Sigma. Genistein, potato acid phosphatase, recombinant (Escherichia coli) protein phosphatase 2B, protease inhibitors, and the PK-C inhibitors GF109203X and calphostin C were from Calbiochem. The PK-C beta II-specific inhibitor LY379196 was a kind gift from Dr. Kirk Ways, Eli Lilly (Indianapolis, IN). Polyvinylidene fluoride (PVDF) membranes (Immobilon P) were from Millipore (Bedford, MA), and Nytran membranes were from Schleicher & Scheull. Radiochemicals and Reflection® autoradiography film were from PerkinElmer Life Sciences. Super Signal chemiluminescence reagent and Iodobeads were from Pierce.

Cell Culture-- THP-1 and HEK 293 cells were obtained from the American Type Culture Collection. Human dermal microvascular endothelial cells and human aortic smooth muscle cells were cultured as described (39, 40). All primary cell cultures used in these studies were of passage 4 or lower.

Measurement of VLDL Receptor Expression by Ligand or Immunoblotting-- Expression of functional VLDL-R was assessed by ligand blotting using RAP or u-PA:PAI-1 complexes as ligands (16, 19). The latter were prepared by incubating two-chain urokinase with active PAI-1 for 30 min at a 1:4 molar ratio. Complex formation was assessed by immunoblotting using anti-u-PA and anti-PAI-1 antibodies, which demonstrated that >80% of the u-PA was incorporated into a u-PA:PAI-1 complex.

For ligand blots, detergent extracts were prepared from control and PMA-treated cells in a buffer containing 25 mM Hepes, pH 7.4, 1% Triton X-100, 0.05% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, and 50 mM D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone). Cellular proteins (100 µg/lane) were separated by 7.5% SDS-PAGE, then transferred to PVDF membranes, which were blocked by incubation in Tris-buffered saline (0.15 M Tris, 0.1 M NaCl, pH 7.4) containing 5% nonfat milk and 0.1% Tween 20. Membranes were then incubated with either 1.0 nM RAP or 2 µg/ml u-PA:PAI-1 complex in the same buffer containing 5 mM CaCl2. After washing, membranes were incubated with 5 µg/ml anti-RAP or anti-PAI-1 antibodies, and bound antibody was detected using peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse IgG. Blots were developed by incubation with Super Signal chemiluminescence reagent.

In selected experiments, the effect of VLDL-R dephosphorylation on ligand binding was assessed. Briefly, extracts of cells that had been exposed to 1.5 × 10-7 M PMA were divided into four aliquots. Proteins within three of these were incubated for 60 min with 0.48, 0.6 or 1.2 units/ml potato acid phosphatase or protein phosphatase 2B, as described previously (41). The fourth aliquot was incubated under identical conditions with an equal volume of phosphate-buffered saline. Extracts were then analyzed by RAP ligand blotting.

For immunoblots, cell extracts were prepared, separated by SDS-PAGE, and transferred to PVDF before incubation with rabbit anti-human VLDL-R antibodies. Membranes were then washed, incubated with peroxidase-conjugated goat anti-rabbit IgG, and developed using chemiluminescence.

Measurement of VLDL Receptor mRNA-- VLDL receptor mRNA was quantitated by Northern blotting. Total RNA was isolated using Trizol (Life Technologies, Inc.) and separated on a 1% denaturing agarose gel. RNA was transferred to nylon membranes in 20× SSC (1× SSC contains 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The membrane was prehybridized at 42 °C in a solution containing 50% formamide, 5× SSC, 5× Denhardt's solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA, and VLDL-R mRNA was detected by overnight hybridization at 42 °C with a full-length VLDL-R cDNA (36) labeled with 32P (Ready to Go labeling kit, Amersham Pharmacia Biotech). To ensure equal loading of RNA in all lanes, blots were stripped and reprobed with a 32P-labeled polymerase chain reaction product corresponding to nucleotides 384-684 of actin cDNA (42).

Binding of VLDL-R Ligands to Control and PMA-treated Cells-- To assess whether PK-C activation altered the expression of VLDL-R on intact cells, cell surface proteins were labeled using sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC biotin) (43) before and after exposure of cells to 1.5 × 10-7 M PMA for 2 h. VLDL-R were then ligand-precipitated from cell extracts using RAP-Sepharose 4B, separated using 7.5% SDS-PAGE, and transferred to PVDF. Biotinylated proteins were detected by chemiluminescence after incubation of membranes with streptavidin-peroxidase.

Two approaches were used to investigate the effect of PK-C activation on the binding of VLDL-R ligands to cells. First, the binding of 125I-RAP to control THP-1 cells or cells incubated for 60 min in the presence of 1.5 × 10-7 M PMA was assessed. 125I-RAP binding was measured by incubating increasing concentrations of 125I-RAP with control or PMA-treated cells (2 × 106 cells/ml, 0.5 ml) for 2 h at 4 °C in the absence or presence of a 100-fold excess of unlabeled RAP. Cells were separated from unbound ligand by centrifugation (10,000 × g) through a 1-ml cushion of silicone oil (Fluid 500:Fluid 200, 7.5:1.5; Dow/Corning, Nye Lubricants, New Bedford, MA), and cell-associated ligand was determined by counting the cell pellets in a gamma  counter. Specific binding was defined as the difference between total and nonspecific binding measured in the absence or presence of excess unlabeled ligand, respectively. Saturation isotherms were prepared from the binding data using non-linear regression and assuming one-site binding (Prism; Graph Pad Software, San Diego, CA) and used to determine the Kd and Bmax. In selected experiments, the role of heparan sulfate proteoglycans in 125I-RAP binding was assessed by measuring binding in the presence of 100 µg/ml of protamine sulfate (44) (Sigma).

In a second series of experiments, the binding of 1,1'-dioctadecyl-3,3',3,3'-tetramethylindocarbocyanine (DiI)-labeled VLDL to control and PMA-treated THP 1 cells was analyzed using flow cytometry. Human VLDL was purified and labeled with DiI as described (45;46). THP-1 cells (2 × 106 cells/ml) were washed and incubated for 2 h at 4 °C with or without 25 µg/ml DiI-labeled VLDL. Cells were then washed again, fixed in 2% paraformaldehyde, and analyzed using a FACScan flow cytometer (Becton-Dickinson; excitation wavelength, 575 nm; emission wavelength, 488 nm) (47). Binding of DiI-VLDL was quantitated by subtracting the mean fluorescence intensity of control cells (autofluorescence) from that of cells incubated with DiI-VLDL. Binding specificity was confirmed by assessing the ability of a 100-fold excess of unlabeled VLDL or RAP to block the binding of DiI-VLDL to cells.

Assessment of the Role of PK-C in Regulation of Receptor Ligand Binding Affinity by PMA-- The role of PK-C in regulation of VLDL-R affinity was assessed by determining the ability of the PK-C inhibitors GF109203X and calphostin C to block PMA-mediated inhibition of VLDL-R ligand binding using both ligand blotting and whole cell binding approaches.

The involvement of the PK-C beta II isoform in mediating the effects of PMA was also assessed. First, we determined whether PK-C beta II was translocated to the membrane fraction after exposure of cells to PMA (48). Next, the ability of the specific PK-C beta II inhibitor LY379196 to block PMA-induced inhibition of VLDL-R ligand binding was measured. Third, the expression of functional VLDL-R by HEK 293 cells transfected with either VLDL-R cDNA alone, or co-transfected with VLDL-R cDNA and a 2980-nucleotide PK-C beta II cDNA (a gift from Dr. George King, Joslin Diabetes Center, Boston, MA) was compared.

Direct Assessment of VLDL Receptor Phosphorylation-- VLDL-R phosphorylation was assessed by measuring incorporation of 32P into the receptor. THP-1 cells (1 × 107 cells/ml) were pre-incubated in phosphate-free RPMI 1640 for 30 min and then divided into 4 aliquots that were each labeled for 3 h with 0.5 mCi/ml [32P]orthophosphate. Sixty minutes before the termination of labeling, either 1.5 × 10-7 M PMA (final concentration), 1.5 × 10-7 M PMA and either 10 µM GF109203X or 30 nM LY379196, or an equal volume of ethanol vehicle was added to the individual aliquots. Labeling was terminated by washing the cells in ice-cold phosphate-buffered saline, and cell pellets were immediately extracted in a buffer containing 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 50 mM D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone and 50 mM sodium fluoride, pH 7.4. Cell extracts were incubated overnight with 30 µl of rabbit anti-VLDL-R antibodies (0.66 mg/ml), and immune complexes were precipitated using 50 µl of protein A-Sepharose. Beads were collected by centrifugation and washed before elution of bound material in 50 µl of 1× Laemmli sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) (49). The precipitate from the cells exposed to PMA alone was divided into two aliquots that were treated for 30 min at 30 °C with either 200 units of recombinant protein phosphatase 2B or potato acid phosphatase or an equal volume of phosphate-buffered saline. Eluates were then separated in parallel lanes of a 7.5% SDS-PAGE gel that was dried and exposed to Reflection autoradiographic film for 24 h at -80 °C. The incorporation of 32P into VLDL-R from each aliquot of cells was determined by measuring the total radioactivity incorporated into the ~130- and ~105-kDa VLDL-R bands using a PhosphorImager (model 445SI; Molecular Dynamics, Sunnyvale, CA). To assure that equal protein synthesis occurred under each of these conditions, a parallel experiment was performed in which aliquots of THP-1 cells were labeled under identical conditions using 0.2 mCi/ml [35S]methionine.

To determine whether 32P was incorporated into serine, threonine, or tyrosine residues, immunoprecipitates from 32P-labeled cells were separated using 7.5% SDS-PAGE, then transferred to PVDF. Membranes were washed in transfer buffer (50 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS, pH 8.3) and then incubated for 1 h at 30 °C in 1.0 M KOH, which causes selective chemical dephosphorylation of phosphoserine residues (50). Immobilized VLDL-R were also analyzed by immunoblotting using specific monoclonal anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine antibodies. Comparative alignment of the cytoplasmic domains of the VLDL-R and low density lipoprotein receptors was performed using the alignment editor function of the PepTools software package (BioTools, Inc., Alberta, CA) with a consensus threshold of 75%.

Preparation of a Truncated VLDL-R Mutant-- To further assess the role of phosphorylation within the VLDL-R cytoplasmic domain on ligand binding activity, a truncated VLDL-R mutant was prepared. Briefly, the 3.2-kilobase VLDL-R cDNA was digested with AseI and BglI to yield 2438, 578, and 238 nucleotide fragments. The gel-purified 2438- and 578-nucleotide fragments were recloned in pRC-CMV to create a construct (pRC-CMV-VLDL-TR) in which the 238-nucleotide cDNA sequence encoding the VLDL-R cytoplasmic domain C-terminal to Leu-790 (proximal to all cytoplasmic domain serine, threonine or tyrosine residues) was deleted. Polymerase chain reaction of the coding region of this construct (sense primer 5'-CTAGTCAACAACCTGAATGA-3', antisense primer 5'-TACACAAGCTTGATCCACGC-3') yielded a 418-nucleotide product, in comparison to a 656-nucleotide product from the wild-type construct. The DNA sequence of the mutant was confirmed by automated sequencing.

HEK 293 cells were transfected with wild type and mutant constructs, and positive clones selected by culture in 400 µg/ml G418. Ligand precipitation of biotinylated cell surface proteins using RAP-Sepharose confirmed that wild type and truncated VLDL-R were expressed in similar amounts on the cell surface.

Effect of Elevated Glucose Concentrations on the Ligand Binding Activity of the VLDL-R-- Elevated glucose concentrations activate endothelial cell PK-C beta II in vitro and in vivo (51, 52), and inhibition of hyperglycemia-induced PKC-beta II activation ameliorated the microvascular complications of diabetes in animal models (53, 54). To determine whether hyperglycemia induced VLDL-R phosphorylation, we incubated human dermal microvascular endothelial cells for 6 h in the following: 1) control media containing 5.5 mM D-glucose, 2) high glucose medium containing 16.5 mM D-glucose, 3) high galactose medium, identical to high glucose medium except for substitution of galactose (16.5 mM) for glucose, and 4) high mannitol medium in which the inert sugar, mannitol (16.5 mM), was substituted for glucose or galactose. Osmolality of all medium was 311 mosmol/kg. Cell extracts were then prepared and separated by 7.5% SDS-PAGE, and the amount of VLDL-R protein and its ligand binding activity were assessed by immunoblotting and RAP ligand blotting, respectively.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Functional VLDL Receptor Expression Is Diminished after Exposure of Cells to PMA-- We initially wished to determine the effect of monocyte differentiation on VLDL-R expression. As a model of monocyte differentiation, THP-1 cells were treated with PMA, which induced them to become adherent and acquire a macrophage-like phenotype (55). Contrary to expectations, the expression of both the ~105- and ~130-kDa VLDL-R isoforms by the PMA-treated cells, measured using RAP ligand blotting, was markedly diminished within 60 min of exposure to PMA (Fig. 1). Maximal responses occurred at a PMA concentration of ~1 × 10-7 M. VLDL-R expression was not altered by treatment of cells with the inactive phorbol ester, 4alpha -phorbol 12,13-didecanoate.



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Fig. 1.   Effect of PMA on the binding of RAP to VLDL-R. THP-1 cells were cultured in the absence (lane 1) or presence (lanes 2-5) of 1.5 × 10-7 M PMA for the indicated times. Detergent extracts were then prepared, and 100 µg of protein from each extract analyzed using RAP ligand blotting, as described under "Experimental Procedures." The ~105-kDa and ~130-kDa bands represent the two isoforms of the VLDL receptor, whereas the 39-kDa band reflects endogenous RAP.

To determine whether these responses were unique to transformed cell lines such as THP-1, we assessed the effects of PMA on the expression of VLDL-R by primary cultures of human dermal microvascular endothelial cells and human aortic smooth muscle cells. As observed with THP-1 cells, exposure to PMA completely blocked the ligand binding activity of VLDL-R from these primary cell cultures as well.

To assess whether these responses were limited to the binding of RAP, which is not a physiological extracellular VLDL-R ligand, we determined the effect of PMA on the binding of u-PA:PAI-1 complexes to the VLDL-R. PMA caused a marked decrease in the binding of these complexes as well (Fig. 2).



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Fig. 2.   Effect of PMA on the binding of u-PA:PAI-1 complexes to VLDL-R. THP-1 cells were cultured for 2 h in the absence (lane 1 of each blot) or presence (lane 2 of each blot) of 1.5 × 10-7 PMA, or PMA and GF109203X (lane 3 of each blot). Detergent extracts from cells incubated under each of these conditions were then divided into equal aliquots and analyzed by ligand blotting using u-PA:PAI-1 complexes as the ligand (A) or immunoblotting, using anti-VLDL-R antibodies (B).

The Effects of PMA Reflect a Decrease in Affinity of the VLDL-R for Its Ligands-- Exposure of THP-1 cells to PMA for 2 h did not affect their content of VLDL-R mRNA (Fig. 3, lanes 1 and 2). Similarly, immunoblotting studies revealed identical amounts of VLDL-R protein within extracts of PMA-treated and control cells (Figs. 2B and 4, upper panel), although markedly less RAP (Fig. 4, lower panel) or u-PA:PAI-1 complex (Fig. 2A) bound to VLDL-R in extracts from the PMA-treated cells. The observations that PMA did not affect the amount of VLDL-R within cell extracts, as determined by immunoblotting, but blocked ligand binding to the receptors suggested that it decreased ligand binding affinity.



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Fig. 3.   Effect of PMA on the levels of VLDL-R mRNA. THP-1 cells were incubated for 2 h in medium containing either no additions (lane 1), 1.5 × 10-7 M PMA (lane 2), 1.0 µM calphostin C (lane 3), or 1.5 × 10-7 M PMA and 1.0 µM calphostin C (lane 4). Total cellular RNA was then isolated, and 10 µg of RNA from cells treated under each of these conditions were analyzed by Northern blotting for VLDL-R (upper panel) or actin mRNA (lower panel).



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Fig. 4.   Effect of PMA on expression of functional VLDL-R and VLDL-R protein. THP-1 cells were incubated for 2 h in medium containing no additions (lane 1 of each blot), 1.5 × 10-7 M PMA (lane 2 of each blot), 10.0 µM GF109203X (lane 3 of each blot), or 1.5 × 10-7 M PMA and 10.0 µM GF109203X (lane 4 of each blot). Cell extracts were then prepared, and equal amounts of protein from each were separated by 7.5% SDS-PAGE and analyzed by RAP ligand blotting (upper panel) or immunoblotting using anti-human VLDL-R antibodies (lower panel).

PMA Inhibits VLDL-R Ligand Binding on Intact Cells-- Two approaches were undertaken to assess the effects of PMA on ligand binding by VLDL receptors on intact cells. First, we compared the binding of 125I-RAP to control and PMA-treated THP-1 cells. 125I-RAP bound specifically to ~31,400 ± 1,038 (S.E.) sites on control cells, with a Kd of ~1.8 ± 0.3 nM, and to ~ 40,110 ± 12,700 (S.E.) sites on PMA-treated cells, with a Kd of ~22.9 ± 4.5 nM (Fig. 5A). Protamine sulfate did not affect the binding of 125I-RAP to either control or PMA-treated cells, suggesting that the observed binding was mediated by the VLDL receptor rather than glycosaminoglycans (56). Consistent with the similar Bmax values determined for the binding of RAP to control and treated cells, PMA did not affect the amount of biotinylated VLDL-R that was immuno- or ligand-precipitated from cell surface-biotinylated THP-1 cells.



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Fig. 5.   Effect of PMA on binding of VLDL-R ligands to intact cells. A, the specific binding of 125I-RAP to THP-1 cells preincubated for 2 h in the absence (------) or presence (----) of 1.5 × 10-7 M PMA was measured as described under "Experimental Procedures." Error bars represent S.E. of triplicate points from a single experiment representative of three so performed. B, specificity of DiI-VLDL binding to THP-1 cells. Histograms represent control THP-1 cells not exposed to PMA or DiI-VLDL (red) and THP-1 cells incubated with DiI-VLDL in the absence (green) or presence (black) of a 100-fold excess of unlabeled VLDL. C, effect of PMA on binding of DiI-VLDL to THP-1 cells. Histograms represent control cells not exposed to PMA or DiI-VLDL (red), cells preincubated for 2 h in the absence (green) or presence (black) of 1.5 × 10-7 M PMA before incubation with 25 µg/ml DiI-VLDL, or cells preincubated for 2 h in the presence of 1.5 × 10-7 M PMA and 10 µM GF109203X (blue) before incubation with DiI-VLDL.

We also examined the effect of PMA on the binding of human VLDL to THP-1 cells. DiI-labeled VLDL (25 µg/ml) bound specifically to control THP-1 cells, as demonstrated by the ability of a 100-fold molar excess of unlabeled VLDL (Fig. 5B) or RAP (not shown) to block the binding of DiI-VLDL to cells by >90%. Exposure of THP-1 cells to PMA decreased the binding of DiI-VLDL by a similar extent as unlabeled VLDL (467.2 to 17.0 arbitrary fluorescence units), with this effect blocked by the PK-C inhibitor GF109203X (Fig. 5C). Taken together, these studies demonstrate that exposure of THP-1 cells to PMA leads to inhibition of the binding of VLDL-R ligands without altering cell surface VLDL-R expression.

The Effects of PMA Depend upon PK-C Activation-- Since PMA activates other protein kinases in addition to PK-C, we further assessed the role of PK-C in the inhibition of VLDL-R ligand binding in response to PMA. In contrast to the protein kinase A inhibitor H89 and the protein-tyrosine kinase inhibitor genistein (not shown), the PK-C inhibitor GF109203X (57) blocked the PMA-induced inhibition of u-PA:PAI-1 complex (Fig. 2A, lane 3), RAP (Fig. 4, upper panel, lane 4), and VLDL binding (Fig. 5C) to the VLDL-R. Similar results were observed with calphostin C, another specific PK-C inhibitor. Neither GF109203X (Fig. 3) nor calphostin C affected the levels of VLDL-R mRNA or protein (Fig. 2B, lane 3; Fig. 4, lower panel, lane 4).

We next assessed the specific involvement of the PK-C beta II isoform in the decreased VLDL-R ligand binding occurring upon PK-C activation. PK-C beta II is strongly expressed in failing human heart (58), a tissue in which we have observed that the VLDL-R is phosphorylated (59). Upon exposure of THP-1 cells to PMA, PK-C beta II was rapidly translocated from the cytosolic to the cell membrane fraction (Fig. 6A). Moreover, as observed with GF109203X, a specific PK-C beta II inhibitor, LY379196, blocked the PMA-induced decrease in RAP binding (Fig. 6B).



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Fig. 6.   Involvement of PK-C beta II in regulation of VLDL-R ligand binding affinity. A, subcellular localization of PK-C beta II (~62 kDa) before and after exposure of THP-1 cells to 1.5 × 10-7 M PMA for 60 min. Cytosolic (upper panel) and membrane (lower panel) fractions were isolated before and at increasing times after exposure of cells to PMA. Proteins from these fractions were separated by 10% SDS-PAGE, transferred to PVDF, and detected by immunoblotting using PK-C beta II-specific antibodies. B, inhibition of the PK-C-associated decrease in VLDL-R ligand binding by the PK-C beta II-specific inhibitor LY379196. THP-1 cells were incubated for 2 h in the absence or presence of 1.5 × 10-7 M PMA or in the presence of PMA and either GF109203X or LY379196. Cell extracts were then prepared and analyzed by RAP ligand blotting.

To further assess the role of PK-C beta II, HEK 293 cells were transfected with VLDL-R cDNA alone or co-transfected with VLDL-R and PK-C beta II cDNA. As previously reported for bovine aortic endothelial cells (60) and mammary carcinoma cells (61, 62), VLDL-R-transfected HEK 293 cells expressed primarily the ~105-kDa isoform of the receptor lacking exon 16, which binds ligands identically to the larger ~130-kDa isoform (2, 62).

In contrast to cells transfected with the VLDL-R alone, co-transfected cells demonstrated a marked increase in the amount of PKC-beta II in the membrane fraction (not shown). Moreover, ligand blot analysis revealed dramatically reduced binding of RAP to immobilized VLDL-R from the co-transfected cells, despite similar amounts of VLDL-R protein (Fig. 7, lane 1 versus lane 5). Moreover, although the PK-C inhibitors GF109203X and LY379196 blocked the inhibitory effects of PMA on the ligand binding activity of the VLDL-R in singly transfected cells, these inhibitors not only blocked the effect of PMA but markedly stimulated VLDL-R ligand binding activity (compared with unstimulated cells) in the co-transfected cells (compare lanes 5 versus lanes 7 and 8). These results are consistent with the increased base-line activity of PK-C beta II in the co-transfected cells.



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Fig. 7.   Transfection of HEK 293 cells with VLDL-R and PKC-beta II cDNA. HEK 293 cells were transfected with a full-length VLDL-R cDNA as described under "Experimental Procedures." Successfully transfected clones were maintained in 400 µg/ml geneticin or co-transfected with a full-length PK-C beta II cDNA. After further selection, the ligand binding activity of VLDL-R from VLDL-R or VLDL-R/PK-C beta II-transfected cell lines was analyzed by ligand blotting (A) and the amount of immunologically detectable receptor quantitated by immunoblotting (B). Compared with VLDL-R from the singly transfected cells, VLDL-R from the co-transfected cells bound less RAP (lane 1 versus lane 5). PMA diminished the ligand binding activity of VLDL-R from single and cotransfectants (lanes 2 and 6). The PK-C inhibitors GF109203X or LY379196 blocked the inhibition of VLDL-R ligand binding activity caused by PMA (lanes 3 and 4), whereas these inhibitors not only blocked the effects of PMA but markedly stimulated the VLDL-R ligand binding activity of the cotransfectants (lanes 7 and 8 versus lane 5).

The VLDL Receptor Is Phosphorylated on Serine Residues-- The results described above suggested that the VLDL-R was phosphorylated in a PK-C-dependent manner, leading to diminished ligand binding affinity. Therefore, we determined whether metabolically labeled THP-1 cells incorporated [32P]orthophosphate into the receptor. In THP-1 cells, 32P was incorporated into both the ~105- and ~130-kDa isoforms of the VLDL-R, with approximately 3-fold more 32P incorporated after exposure to PMA. Inclusion of GF109203X or LY379196 during metabolic labeling or treatment of the immunoprecipitated VLDL-R with protein phosphatase 2B reduced receptor phosphorylation to base-line levels (Fig. 8A). In control studies performed in parallel, PMA did not affect the levels of [35S]methionine-labeled VLDL-R (Fig. 8C), suggesting that it did not influence receptor synthesis or degradation. Immunoblotting of immunoprecipitated VLDL-R using anti-VLDL-R antibodies confirmed that similar amounts of VLDL-R were immunoprecipitated from control and PMA-treated cells (Fig. 8D).



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Fig. 8.   Incorporation of 32P into the VLDL-R. A, THP-1 cells were metabolically labeled by incubation for 3 h in phosphate-free medium containing 0.5 mCi/ml of [32P]orthophosphate. During the last 60 min of the labeling period, either ethanol control (lane 1), 1.5 × 10-7 M PMA (lane 2), 1.5 × 10-7 M PMA and 10 µM GF109203X (lane 3), or 1.5 × 10-7 M PMA and 30 nM LY379196 (lane 4) were added to the cell suspensions. An aliquot from the cells exposed to PMA was also treated with recombinant protein phosphatase 2B after labeling (lane 5). Detergent extracts were prepared, and VLDL receptors were immunoprecipitated using anti-VLDL-R antibodies and protein A-Sepharose. Precipitated VLDL-R were eluted with 50 µl of 1× Laemmli buffer, and proteins within equal volumes of eluate were separated using 7.5% SDS-PAGE, transferred to PVDF, and detected using autoradiography. B, the PVDF membranes used to prepare the autoradiogram in panel A were incubated in 1 M KOH for 1 h, and autoradiograms were repeated. C, THP-1 cells were metabolically labeled with [35S]methionine under identical conditions as in panel A, and VLDL-R were then immunoprecipitated and analyzed as in panel A. D, immunoblot analysis, using anti-VLDL-R antibodies, of the immunoprecipitated VLDL-R depicted in panels A and B. U, units.

Incubation of PVDF-immobilized 32P-labeled VLDL-R with 1 M KOH, which chemically dephosphorylates phosphoserine residues (50), released 32P from the receptor (Fig. 8B). The specificity of this approach was confirmed by the observation that immobilized phospho-MAP kinase (tyrosine-phosphorylated, from vascular endothelial cell growth factor-stimulated human dermal microvascular endothelial cells) was not dephosphorylated when subjected to identical treatment (not shown). Moreover, VLDL-R immunoprecipitated from extracts of PMA-treated THP-1 cells were recognized by anti-phosphoserine but not anti-phosphothreonine or anti-phosphotyrosine antibodies.

Direct Evidence for Modulation of VLDL Receptor Ligand Binding Affinity by Phosphorylation-- Since the studies above demonstrated that the VLDL-R was phosphorylated in intact cells and that receptor phosphorylation diminished ligand binding affinity, we determined whether the affinity of the receptor for RAP was restored after enzymatic dephosphorylation. Treatment of extracts from PMA-treated THP-1 cells (which do not bind RAP) with potato acid phosphatase restored the binding of RAP to immobilized VLDL-R in a concentration-dependent manner (Fig. 9, lanes 3-5). An identical effect was observed after treatment of the phosphorylated receptor with protein phosphatase 2B.



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Fig. 9.   Effect of VLDL-R dephosphorylation on RAP binding activity. Detergent extracts were prepared from either untreated THP-1 cells (lane 1) or cells exposed to 1.5 × 10-7 M PMA for 2 h (lane 2). Extracts from the PMA-treated cells, containing 100 µg of total protein, were incubated with either phosphate-buffered saline (lane 2) or increasing amounts of potato acid phosphatase at 30 °C for 60 min (lanes 3-5) and analyzed by RAP ligand blotting. Similar results were obtained using recombinant protein phosphatase 2B.

Additional evidence for a critical role for phosphorylation within the VLDL-R cytoplasmic domain in regulation of receptor ligand binding affinity was derived from study of a VLDL-R mutant lacking the cytoplasmic domain of the receptor. This mutant receptor was expressed in similar amount as the wild-type receptor (primarily as the ~105-kDa isoform) on the surface of transfected HEK 293 cells, as determined by analysis of immunoprecipitated VLDL-R from cell surface-biotinylated cells. However, unlike the wild-type receptor, binding of RAP by the truncated receptor was unaffected after treatment of cells with PMA (Fig. 10). Taken together with the preceding studies, these experiments support the hypothesis that phosphorylation of serine residues within the cytoplasmic domain of the receptor plays an important role in regulating ligand binding affinity.



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Fig. 10.   PK-C independence of a truncated VLDL-R mutant. Stable transfectants expressing wild-type VLDL-R (panel A) or a truncated VLDL-R mutant lacking the cytoplasmic domain (panel B) were prepared in HEK 293 cells. Each of the transfectants were incubated in the absence (lane 1) or presence (lane 2) of 1.5 × 10-7 M PMA or PMA and either 10 µM GF109203X (lane 3) or LY379196 (lane 4) for 2 h. Detergent extracts were then prepared and analyzed by RAP ligand blotting. PMA diminished the ligand binding activity of the wild-type receptor (panel A, lanes 1 versus 2), with its effects prevented by PK-C (beta II) inhibition (lanes 3 and 4), but did not affect that of the truncated receptor (panel B).

Activation of PK-C by Elevated Glucose Concentrations Leads to Inhibition of VLDL-R Ligand Binding Activity-- Elevated glucose concentrations contribute to the pathogenesis of vascular disease in diabetes mellitus (52, 63). Culture of microvascular endothelial cells in the presence of high glucose or galactose stimulates the activation of PK-C by inducing de novo synthesis of diacylglycerol (51, 64). Treatment of diabetic rats with a specific PK-C beta II inhibitor inhibits the development of vascular dysfunction that leads to retinal, renal, and neurologic disease (53, 54, 65).

To determine whether the ligand binding activity of the VLDL receptor was affected by high glucose, microvascular endothelial cells were incubated in the presence of various concentrations of glucose, galactose, or mannitol. Exposure of cells to 16.5 mM D-glucose or galactose caused a potent and rapid reduction in the binding of RAP by VLDL-R (Fig. 11, upper panel), whereas the level of VLDL-R detected by immunoblotting was unaffected (Fig. 11, lower panel). The effects of glucose and galactose were blocked by GF109203X and LY379196 (Fig. 11, upper panel, lanes 3 and 4 and 6 and 7, respectively), demonstrating an important role for PK-C, particularly the beta II isoform, in this response. An identical concentration of mannitol, which does not induce diacylglycerol synthesis or PK-C beta II activation in endothelial cells (51), did not affect the binding of RAP by VLDL-R.



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Fig. 11.   Effect of hyperglycemia on VLDL-R ligand binding activity; PK-C dependence. Human dermal microvascular endothelial cells were cultured for 6 h in control medium (lane 1) or medium containing elevated concentrations of glucose, galactose, or mannitol in the absence or presence of GF109203X or LY379196 (lanes 2-8). Detergent extracts were then prepared, and VLDL-R ligand binding activity was assessed using RAP ligand blotting (upper panel). Elevated concentrations of glucose (lane 2) or galactose (lane 5) inhibited VLDL-R ligand binding activity, with these effects blocked by GF109203X (lanes 3 and 6) or LY379196 (lanes 4 and 7). Elevated concentrations of mannitol (lane 8) did not affect VLDL-R ligand binding activity. The amount of VLDL-R protein, as determined by immunoblotting using anti-VLDL-R antibodies (lower panel), was unaffected by any of these treatments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that PK-C activation leads to phosphorylation of the VLDL-R and inhibition of its ligand binding activity in a number of cell types, including monocyte-derived THP-1 cells, human endothelial and vascular smooth muscle cells, and HEK 293 cells transfected with VLDL-R cDNA. PK-C-dependent phosphorylation of the VLDL-R leads to diminished binding not only of RAP but of other VLDL-R ligands such as u-PA:PAI-1 complexes and VLDL. The (patho)physiological relevance of this process is supported by the observation that glucose concentrations similar to those that induce PK-C activation in vitro and in vivo in experimental diabetes cause VLDL-R phosphorylation and loss of ligand binding activity in endothelial cells. Moreover, VLDL-R isolated from myocardium obtained from patients with advanced cardiomyopathies, a condition associated with PK-C beta II activation in the heart (58), are also phosphorylated, whereas receptors from normal human myocardium are not.2

VLDL-R phosphorylation was associated with a rapid loss of ligand binding activity, although levels of VLDL-R mRNA and protein and the expression of the VLDL-R on the cell surface were unaffected. Taken together, these observations suggest that phosphorylation results in diminished affinity of the receptor for its ligands. This conclusion is supported by direct radioligand binding studies in which 125I-RAP was found to bind to a similar number of sites on control and PMA-treated THP-1 cells, although with ~13-fold lower affinity to the latter. Furthermore, although the affinity of DiI-VLDL binding to THP-1 cells was not determined directly, the complete disappearance of binding after exposure of these cells to PMA suggests that VLDL-R phosphorylation greatly diminishes the affinity of the receptor for apoE-containing ligands as well.

Several observations support the conclusion that phosphorylation of cytoplasmic domain serine residue(s) regulates the ligand binding activity of the VLDL-R through a process involving PK-C. First, the PMA-induced loss of ligand binding activity was prevented by the PK-C inhibitors GF109203X (57), calphostin C (66), and LY379196 but not by inhibitors of PK-A or tyrosine kinases. Second, enzymatic dephosphorylation of the VLDL-R restored its ligand binding activity. Third, 32P was directly incorporated into the VLDL-R, with incorporation stimulated by PMA. Fourth, chemical dephosphorylation of the VLDL-R, as well as immunoblotting with specific monoclonal antibodies, were consistent with the phosphorylation of serine but not threonine or tyrosine residues within the VLDL-R in response to PK-C activation. Finally, the ligand binding activity of a mutant VLDL-R lacking the cytoplasmic domain was unaffected by PMA.

The effects of phosphorylation on VLDL receptor activity likely reflect a generalized mechanism through which phosphorylation within a receptor cytoplasmic domain regulates its function by altering ligand binding affinity. For example, phosphorylation of serine and threonine residues within the cytoplasmic domains of the insulin (67, 68) and epidermal growth factor receptors (69-75) diminishes their affinity for insulin and epidermal growth factor, respectively. With respect to the epidermal growth factor receptor, these effects mimic those involved in regulation of the receptor by heterologous ligands (76) such as platelet-derived growth factor (77) and those induced through a negative feedback pathway triggered by binding of epidermal growth factor itself (78).

Regulation of the activity of LDL receptor family members through phosphorylation is a novel concept of which few examples exist. Though Kishimoto et al. (79) observe that the LDL receptor could be phosphorylated on Ser833 (79) by an LDL receptor kinase purified from bovine adrenal cortex (80), these effects occurred only in a cell-free system, and incorporation of [32P]orthophosphate into the LDL receptors of intact cells could not be demonstrated (80). More recently, however, Bu et al. (81) demonstrate that the LDL receptor-related protein/alpha 2-macroglobulin receptor is phosphorylated in neuronal cells in response to nerve growth factor, leading to enhanced expression and endocytic activity. Our studies suggest that the cytoplasmic domain of the VLDL-R plays an important role in regulating receptor function, a hypothesis consistent with the complete sequence conservation of this domain in the murine (38, 82), rabbit (6), and human (35-37) receptors. Trommsdorf et al. (83) also demonstrate a critical role for the cytoplasmic domains of the VLDL-R and apoE receptor 2 in binding of the cytoplasmic adaptor protein mammalian Disabled (mDab1), an interaction critical for appropriate migration and layering of neurons during development of the cerebellum and cerebral cortex. Whether this process may be affected by VLDL-R phosphorylation has not been assessed.

Which serine residue(s) within the cytoplasmic domain of the VLDL-R are involved in regulation of ligand binding affinity is uncertain, although preliminary studies suggest the involvement of Ser-829.3 Interestingly, although the cytoplasmic domains of the VLDL and LDL receptors are highly homologous (49% matching residues), only 2 of the 6 serines within this region of the VLDL-R align with any of the 4 serines in the corresponding domain of the LDL-R, perhaps explaining the different susceptibility of these receptors to phosphorylation in intact cells.

In addition to hyperglycemia, PK-C activation may be induced by several cytokines that affect the function of endothelial and smooth muscle cells and monocytes (84-86). Mechanical stimuli (87, 88) and hypoxia (89) also activate PK-C. The pathophysiologic consequences of excessive PK-C beta II activation in myocardium, as occurs in diabetes (63, 65) and congestive cardiomyopathies (58), is supported by transgenic mouse models in which myocardial PKC beta II overexpression leads to a fatal postnatal cardiomyopathy (90). Although our studies demonstrate the involvement of PK-C beta II in phosphorylation of the VLDL-R, however, they do not exclude the possibility that other PK-C isoforms such as PK-C delta  may also mediate this activity (91).

Our studies must be considered in light of the fact that homozygous disruption of the VLDL-R gene is not associated with major phenotypic abnormalities (92). Although these results demonstrate that the VLDL receptor does not play a critical role in lipoprotein clearance in mice, they do not exclude a role for this receptor in normal and/or pathophysiological processes. The ability of the VLDL-R to mediate lipoprotein uptake in vivo has been demonstrated by gene transfer studies (93, 94), and the receptor may be involved in other processes in addition to lipoprotein metabolism. Hence, a complementary interpretation of the VLDL receptor knockout studies, based on the findings presented here, is that in at least some tissues the receptor may be phosphorylated and in a relatively inactive state. If so, then genetic deletion of the receptor might have relatively minor consequences. Further evaluation of this hypothesis will require in vivo examination of the function of VLDL-R lacking specific serine residues involved in regulation of receptor affinity.


    FOOTNOTES

* This work was supported by National Institute of Health Grants HL50827 (to K. R. M.) and HL50787 (to D. K. S.) and a Research Award from the American Diabetes Association (to K. R. M.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A recipient of a Scientist Development Grant from the American Heart Association.

A recipient of a postdoctoral fellowship from the Ohio Valley affiliate of the American Heart Association.

Dagger Dagger To whom correspondence should be addressed: Hematology-Oncology Division, BRB 329, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-6606; Fax: 216-368-1166; E-mail: kxm71@po.cwru.edu.

Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M003953200

2 R. Sakthivel, manuscript in preparation.

3 R. Sakthivel and K. McCrae, unpublished data.


    ABBREVIATIONS

The abbreviations used are: VLDL, very low density lipoprotein receptor; LDL-R, low density lipoprotein associated; PMA, phorbol 12-myristate 13-acetate; RAP, LDL receptor-associated protein/alpha 2-macroglobulin receptor-associated protein; PK-C, protein kinase C; u-PA, two-chain urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor type 1; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; DiI, 1,1'-dioctadecyl-3,3',3,3'-tetramethylindocarbocyanine.


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