©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Glycoprotein 330 as an Endocytic Receptor for Apolipoprotein J/Clusterin (*)

Maria Z. Kounnas (1)(§), Elena B. Loukinova (1), Steingrimur Stefansson (1), Judith A. K. Harmony (2)(¶), Bryan H. Brewer (3), Dudley K. Strickland (1), W. Scott Argraves (1)(**)

From the (1) J. H. Holland Laboratory, Biochemistry Department American Red Cross, Rockville, Maryland 20855, the (2) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, and the (3) Molecular Diseases Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (K= 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.


INTRODUCTION

The endocytic receptor gp330() is expressed by many specialized epithelial cells such as those of renal proximal tubules, ependyma, lung alveoli, and mammary gland epithelium (Kerjaschki and Farquhar, 1983; Zheng et. al.; 1994, Kounnas et al., 1994). gp330 is typically found on the apical surfaces of these epithelia, which are exposed to fluid-filled spaces. It is believed that gp330 functions to mediate endocytosis of ligands based on its cytoplasmic domain having consensus endocytosis signal motifs (Raychowdhury et al., 1989; Saito et al., 1994), that it has been localized within clathrin-coated pits and vesicles (Kerjaschki and Farquhar, 1983; Kerjaschki et al., 1987), and that it is structurally related to a family of receptors including the low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP) (Raychowdhury et al., 1989; Saito et al., 1994; Korenberg et al., 1994) that mediate cellular internalization of their ligands.

Efforts to identify ligands for gp330 have revealed that gp330, like LRP, is capable of binding in vitro to a number of different ligands including RAP, a 39-kDa protein that copurifies with gp330 and LRP (Kounnas et al., 1992b; Orlando et al., 1992; Christensen et al., 1992), lipoprotein lipase (Willnow et al., 1992; Kounnas et al., 1993), apolipoprotein E-enriched - very low density lipoproteins (Willnow et al., 1992), complexes of tissue-type plasminogen activator and plasminogen activator inhibitor-1 (tPA:PAI-1) (Willnow et al., 1992), and complexes of urokinase plasminogen activator and PAI-1 (uPA:PAI-1) (Moestrup et al., 1993b). Each of the proteins that bind to gp330 have been shown to also bind LRP. However, LRP binds several additional ligands, such as -macroglobulin-protease complexes (Ashcom et al., 1990) and Pseudomonas aeruginosa exotoxin A (Kounnas et al., 1992a) that are not shared with gp330. To date no ligand has been identified that can bind gp330 exclusively.

While the overlapping ligand binding specificity of LRP and gp330 might suggest that gp330 is part of a redundant receptor system, the very different pattern of expression of the two receptors suggests that gp330 has a unique function with respect to LRP. Not only is gp330 expression much more restricted than is LRP (Zheng et al., 1994; Kounnas et al., 1994), but in those cells where both receptors are expressed the two proteins have distinct subcellular distribution patterns (Kounnas et al., 1994). gp330 is strictly confined to apical portions of cells within epithelial layers, whereas LRP seems to be more basolaterally distributed within gp330-expressing cells. In addition, gp330 can be found expressed exclusive of LRP such as in the epithelial cells of epicardium and renal proximal tubules.

In an effort to understand the function of gp330 we have sought to identify gp330 ligands present in bodily fluids that are normally in contact with gp330-expressing epithelial cells. Given that gp330 has been shown to be expressed by mammary epithelial cells (Zheng et al., 1994), we have examined milk as a source for gp330 ligands. Herein we report on the identification of a milk protein known as apolipoprotein J (apoJ)() as a novel gp330 ligand. Furthermore we have used gp330-expressing cell lines to demonstrate that gp330 mediates internalization and degradation of apoJ.


MATERIALS AND METHODS

Proteins

gp330 from human urine or porcine kidney brush border membrane extracts was isolated by RAP-Sepharose affinity chromatography as described previously (Kounnas et al., 1992b, 1993). LRP was isolated from human placenta by -macroglobulin/methylamine-Sepharose affinity chromatography as described previously (Ashcom et al., 1990). Human RAP, expressed as a glutathione S-transferase fusion protein in bacteria, was prepared (free of glutathione S-transferase) as outlined by Williams et al. (1992). ApoJ was isolated from human plasma by immunoaffinity chromatography and reverse phase high performance liquid chromatography (de Silva et al., 1990b). Apolipoprotein E3 (apoE3) was purified as described by Kelly et al.(1994). Pro-urokinase (pro-uPA) and uPA were provided by Dr. Jack Henkin (Abbott Laboratories, Abbott Park, IL). PAI-1 was purchased from Molecular Innovations (Royal Oak, MI). Human lactoferrin was provided by Dr. David Mann (American Red Cross, J. H. Holland Laboratory, Rockville, MD). The 18-kDa carboxyl-terminal fragment of human lipoprotein lipase (residues 313-448) was from Dr. David Chappell (University of Iowa College of Medicine, Iowa City, IA). uPA:PAI-1 complexes were generated by incubation of a 1:1 molar ratio of active uPA with active PAI-1 for 30 min at room temperature. Bovine serum albumin-fraction V, and ovalbumin were purchased from Sigma.

Antibodies

The mouse monoclonal antibody to human apoJ designated 1D11 has been described previously (de Silva et al., 1990a). The mouse monoclonal antibody to rat gp330 designated 1H2 was provided by Dr. Robert McCluskey (Harvard/Massachusetts General Hospital, Boston, MA). This antibody has previously been shown to cross react to both porcine (data not shown) and human gp330 (Kounnas et al., 1993). The mouse monoclonal antibody to the 515-kDa heavy chain of human LRP designated 8G1 has been described previously (Strickland et al., 1991). IgG was purified on protein G-Sepharose from each of the mouse ascitic fluids. IgG was isolated from rabbit polyclonal anti-gp330 serum (rb239) (Kounnas et al., 1994) and anti-LRP serum (rb777 and rb810) (Strickland et al., 1991) by affinity chromatography on either gp330- or LRP-Sepharose (1-2 mg of protein/ml of resin) followed by absorption on RAP-Sepharose.

gp330-Sepharose Affinity Chromatography of Human Breast Milk

An affinity matrix of gp330-Sepharose was prepared by coupling porcine gp330 to activated CNBr-activated Sepharose (Pharmacia Biotech Inc.) at 1-2 mg of protein/ml of resin. Human breast milk (12-20 weeks postdelivery) was centrifuged for 1 h at 100,000 g in the presence of the following proteinase inhibitors: phenylmethylsulfonyl fluoride (1 mM), leupeptin (4 µg/ml), pepstatin (66 ng/ml), and D-Phe-Pro-Arg-CHCl (4 µg/ml). After centrifugation, the milk supernatant (200 ml) was preabsorbed on CL-4B Sepharose (20 ml bed volume) and applied to the gp330-Sepharose affinity matrix (5 ml bed volume). The column was then washed with 50 volumes of 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS), and bound protein was eluted with 2 column volumes of 8 M urea, 50 mM Tris, pH 7.4. Eluted fractions were electrophoresed in the presence of SDS on 4-12% polyacrylamide gradient gels (Novex, San Diego, CA). Protein bands were excised from SDS-PAGE gels, extracted in 0.1% SDS, 0.1 M Tris, pH 8.0, and sequenced on a Hewlett Packard (model Gl000S) protein sequencing instrument.

Solid Phase Binding Assays

Enzyme-linked immunosorbent assays (ELISA) were carried out as described previously (Kounnas et al., 1993) except that 0.5% ovalbumin was used in the blocking buffer instead of bovine serum albumin. ELISA data was analyzed using a form of the binding isotherm as described by Ashcom et al.(1990). Both homologous and heterologous ligand displacement assays were conducted according to methods already outlined (Williams et al., 1992). Briefly, microtiter wells were coated with gp330, LRP, or ovalbumin (5 µg/ml) in TBS, 5 mM CaCl, pH 8.0, for 4 h at 37 °C, and nonspecific sites were blocked with 0.5% ovalbumin, TBS. Receptor-coated wells were then incubated with radiolabeled ligand in 0.5% ovalbumin, TBS, 5 mM CaCl, 0.05% Tween-20 in the presence of unlabeled competitor at varying concentrations for 18 h at 4 °C. For binding assays, apoJ was labeled with I using IODO-GEN (Pierce Chemical Co., Rockford, IL) and used within 24 h at a concentration of 0.5-2 nM (specific activity ranged from 1 to 5 mCi/mg of protein). Binding data was analyzed, and dissociation constants (K ) and inhibition constants (K ) were determined using the computer program LIGAND (Munson and Rodbard, 1980).

Gel Blot Overlay Assays

ApoJ and RAP (0.5 µg each) were electrophoresed in the presence of SDS and under nonreducing conditions on 4-12% polyacrylamide gradient gels (Novex, San Diego, CA) and then transferred to nitrocellulose membranes and blocked with 3% nonfat milk, which lacks detectable levels of apoJ (data not shown), in TBS (blocking buffer). The membranes were then incubated with unlabeled gp330 or LRP (25 nM) in blocking buffer with 5 mM CaCl and 0.05% Tween-20. After a 1-h incubation, the membranes were washed with TBS, 0.05% Tween-20 and then probed with I-labeled monoclonal antibodies to gp330 (1H2) or LRP (8G1) (0.1 nM) for 1 h at 25 °C. Filters were washed and exposed to Kodak X-Omat AR film (Eastman Kodak Co.) at -70 °C.

Cell-mediated Ligand Internalization and Degradation Assays

Cell assays were carried out according to procedures described previously (Kounnas et al., 1993). Mouse embryonal carcinoma F9 cells (ATCC CRL1720) were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Inc.) in tissue culture plates (Corning, Corning, NY) precoated with 0.1% gelatin. To augment expression of gp330, the F9 cells were induced to differentiate by treatment with 0.1 µM retinoic acid (Calbiochem, San Diego, CA) and 0.2 µM dibutyryl cAMP (DBC, Sigma, St. Louis, MO) in complete Dulbecco's modified Eagle's medium for 7 days. After 7 days of treatment, the cells were transferred to gelatin-coated 6-well dishes at 3-5 10 cells/well (60-80% confluence) and allowed to grow for 24 h. Prior to the addition of radioactive ligands, the cells were washed and incubated in Dulbecco's modified Eagle's medium containing 20 mM HEPES, Nutridoma serum substitute (Boehringer Mannheim), penicillin/streptomycin, and 1.5% bovine serum albumin (assay medium) for 1 h at 37 °C. Radioiodinated apoJ (5-10 nM) in assay medium was added to the cells in the presence or absence of increasing concentrations of RAP or unlabeled apoJ (1.8-450 nM) and incubated for 18 h at 37 °C. Typically, 200 µg of apoJ was radioiodinated using IODO-GEN (specific activity of 1-5 mCi/mg protein) and used within 24 h. For assays evaluating the effect of gp330 antibodies on I-apoJ internalization and degradation, the cells were preincubated with antibodies to gp330 (rb239) or antibodies to LRP (rb777) for 1 h at 37 °C prior to the addition of I-apoJ. Antibodies were also included during the subsequent incubation with labeled ligand. Radioactivity in the cell medium that was soluble in 10% trichloroacetic acid was taken to represent degraded ligand (Goldstein and Brown, 1974). Total ligand degradation was corrected for any degradation that occurred in radioligand-containing medium lacking cells, typically this amount was 5-10% of the total counts added. To inhibit lysosomal protease activity, the cells were treated with 0.1 mM chloroquine (Sigma) throughout the duration of the internalization and degradation assays. To determine the amount of I-apoJ that was internalized, the cells were washed 3 times with isotonic phosphate-buffered saline and then treated with serum-free medium containing 0.5 mg/ml trypsin, 0.5 mg/ml proteinase K (Sigma), and 0.5 mM EDTA for 2-4 min at 4 °C. The washing and trypsin/proteinase K/EDTA treatment were found to remove >80% of the surface-associated I-apoJ (data not shown). The cells were then centrifuged at 6000 g for 2 min, and the amount of radioactivity in the cell pellet was measured.


RESULTS

gp330-Sepharose Chromatography of Human Milk Selects ApoJ

The fact that gp330 expression in vivo is restricted to epithelial cells in contact with fluids prompted us to examine biological fluids for the presence of gp330 ligands. Human breast milk was available in larger quantities than other potential gp330 ligand-containing fluids like cerebral spinal fluid and seminal plasma. An affinity matrix of gp330 coupled to Sepharose was used to select gp330-binding proteins from human skim milk. The skim milk was preabsorbed on a column of Sepharose and then applied to a column of gp330-Sepharose. Elution of the gp330-Sepharose column with a buffer containing urea released a polypeptide that when analyzed by SDS-PAGE had a molecular mass of 70 kDa (Fig. 1). The 70-kDa protein was subjected to protein sequence analysis, which revealed two sequences identical to the first 10 residues of the - and -subunits of human apolipoprotein J, a known 70-kDa milk protein (de Silva et al., 1990a, 1990b). In immunoblotting analysis, monoclonal apoJ antibodies were found to react with the 70-kDa protein eluted from the gp330 affinity matrix (data not shown). The results show that apoJ can be isolated from milk by gp330-Sepharose affinity chromatography and suggest that gp330 interacts with apoJ.


Figure 1: Selection of apoJ by gp330-Sepharose affinity chromatography. Human skim milk was passed over gp330-Sepharose, the column was washed with 50 mM Tris, 0.15 M NaCl, pH 8.0, and the bound fractions were eluted with 2 column volumes of 8 M urea, 50 mM Tris, pH 7.4. Lanes 1-6 represent SDS-PAGE analysis of sequentially eluted fractions from the affinity column stained with Coomassie Blue. The migration positions of molecular mass standards are indicated on the right in kDa.



ApoJ Binds with High Affinity to gp330 but Not to LRP

We next investigated the ability of apoJ to bind to gp330 by using several types of solid-phase binding assays. In ELISA, apoJ bound to gp330 (urine or kidney derived) immobilized on microtiter well plastic with half-saturating levels of binding achieved at 2.8 nM for porcine gp330 and 3.8 nM for human gp330 (Fig. 2). Homologous ligand competition assays were performed to obtain quantitative information regarding the apoJ-gp330 interaction. As shown in Fig. 3A, I-apoJ bound to immobilized gp330, and the binding could be inhibited with increasing concentrations of unlabeled apoJ. The binding data could be fit to a model containing a single class of sites with a K of 10 nM. In these assays, we also investigated the ability of apoJ to bind to LRP, a receptor closely related to gp330 in structure and function. However, no binding of I-apoJ to immobilized LRP was observed (Fig. 3A). In addition, no binding was observed between apoJ and LRP in ELISA (data not shown). The functionality of the LRP used in the assays was confirmed by its ability to promote binding of I-apoE, a known LRP ligand (Beisiegel et al., 1989; Kowal et al., 1989) (Fig. 3B). In these experiments I-apoE also bound to gp330 with a K of 50.7 nM, which was similar to that for LRP, K= 53.6 nM. The apoJ-gp330 interaction was also evaluated by using a gel blot-overlay assay in which apoJ, transferred onto nitrocellulose filters after SDS-PAGE, was incubated with buffer containing either gp330 or LRP. Bound receptor was then detected by using radiolabeled monoclonal receptor antibodies. As shown in Fig. 4A, lane 4, gp330 bound to immobilized apoJ, whereas LRP did not (Fig. 4B, lane 4). A control for the functionality of both receptors in this assay was their ability to bind to the 39-kDa protein RAP (Fig. 4, lane 2 in panels A and B). The results of both the solid phase microtiter well assays and gel blot overlay assays show that apoJ binds to gp330 but not to LRP.


Figure 2: Binding of apoJ to gp330 as measured by ELISA. Increasing concentrations of apoJ were incubated with microtiter wells coated with human gp330 (), porcine gp330 (), or ovalbumin () (5 µg/ml). Bound apoJ was detected with the monoclonal anti-apoJ IgG 1D11. The half-saturation values determined for porcine and human gp330 were 2.8 and 3.8 nM, respectively. The data presented are representative of three experiments each performed in duplicate.




Figure 3: Binding of I-apoJ and I-apoE3 to gp330. In panel A, I-labeled apoJ (1 nM) was incubated for 18 h at 4 °C in wells coated with gp330 (), LRP (), or ovalbumin () in the presence of increasing concentrations of unlabeled apoJ (0.2-450 nM). After incubation, wells were washed and counted. The data were analyzed by the program LIGAND, and the curve represents the best fit of the data to a single class of sites with a K of 10 nM. The data presented are representative of six experiments having a mean K = 14.2 ± 8.6 nM). In panel B, I-labeled apoE3 (2 nM) was incubated with gp330, LRP, or ovalbumin-coated wells in the presence of increasing concentrations of unlabeled apoE3 (0.2-450 nM) for 18 h at 4 °C. The K values measured for the binding of apoE3 to gp330 and LRP were 50.7 and 53.6 nM, respectively.




Figure 4: I-gp330 binding to apoJ immobilized on nitrocellulose membranes after SDS-PAGE. ApoJ (0.5 µg, lane 2) or RAP (0.5 µg, lane 4) were electrophoresed on 4-12% polyacrylamide gradient gels under nonreducing conditions and then transferred to nitrocellulose membranes. In panel A, the membrane was probed with unlabeled gp330 (25 nM), and the bound receptor was detected with I-labeled monoclonal anti-gp330 IgG 1H2. In panelB, the membrane was probed with unlabeled LRP (25 nM), and bound receptor was detected with I-labeled monoclonal anti-LRP IgG 8G1. Both membranes were washed and exposed to x-ray film. The migration positions of molecular mass standards are indicated on the right in kDa.



The gp330 Ligands RAP, ApoE3, and Lipoprotein Lipase Carboxyl-terminal Receptor-binding Fragment Block Binding of ApoJ to gp330

We next evaluated the ability of several previously described gp330 ligands to inhibit the binding of apoJ to the receptor. Heterologous ligand competition assays were performed using RAP, apoE3, or lipoprotein lipase carboxyl-terminal receptor-binding fragment as competitors for radiolabeled apoJ binding to gp330-coated microtiter wells. As shown in Fig. 5, each of these ligands competed effectively for the I-apoJ binding (Fig. 5). By curve fitting the data, inhibition of binding constants (K ) were determined for each ligand (). Inhibition constants were also derived for several other gp330 ligands including pro-uPA, uPA:PAI-1, and lactoferrin, and the values are shown in (binding curves not shown). While apoJ, RAP, apoE, and lipoprotein lipase carboxyl-terminal receptor-binding fragment were able to compete for I-apoJ binding to gp330, the other gp330 ligands pro-uPA, uPA:PAI-1, and lactoferrin were not able to inhibit the binding. The results indicate that apoJ, apoE3, and lipoprotein lipase carboxyl-terminal receptor-binding fragment may share a common binding site on gp330 or have binding sites in close proximity to one another, whereas the other ligands bind to separate sites on the receptor. Similar results and interpretations have been reported for the binding of many of these ligands to LRP (Moestrup et al., 1993a).


Figure 5: The binding of I-apoJ to gp330 can be inhibited by RAP, lipoprotein lipase carboxyl-terminal receptor-binding fragment, and apoE3. I-labeled apoJ (1 nM) was incubated overnight at 4 °C in wells coated with gp330 (5 µg/ml) in the presence of increasing concentrations of unlabeled apoJ (), RAP (), lipoprotein lipase carboxyl-terminal receptor-binding fragment (), or apoE3 (). Following incubation, the wells were washed, and bound radioactivity was measured. The binding data was analyzed using the program LIGAND. The data presented are representative of two experiments, each performed in duplicate. The curves represent the best fit data for a single class of sites, and the derived inhibition constants (K) are indicated in Table I. The binding of I-apoJ, in the presence of increasing concentration of unlabeled apoJ, to wells coated with ovalbumin is shown as a control ().



gp330 Mediates the Internalization of ApoJ in F9 Cells

We studied the uptake of I-apoJ by gp330-expressing embryonal carcinoma F9 cells in order to determine if gp330 could mediate its cellular endocytosis and degradation. The F9 cells were grown in the presence of RA and DBC since this treatment can increase gp330 expression levels 20-40-fold (Fig. 6, inset) while having no effect on LRP, very low density lipoprotein receptor, or LDLR levels.() As shown in Fig. 6, RA/DBC-treated F9 cells internalized 17-fold greater levels of I-gp330 monoclonal antibody (1H2) than untreated cells. Similar results were observed with I-gp330 polyclonal antibodies (data not shown). The increased ability of the RA/DBC-treated cells to endocytose gp330 antibody is comparable in magnitude to the increase in gp330 protein levels in the treated cells as measured by immunoblotting analysis of F9 cell extracts (Fig. 6, inset). Both the untreated and RA/DBC-treated F9 cells were found to internalize and degrade I-apoJ (Fig. 7). The levels of uptake and degradation of I-apoJ were respectively 5- and 10-fold greater in the RA/DBC-treated F9 cells as compared with the untreated cells. Several cells lines that do not express gp330 but are known to express LRP such as HepG2 and Chinese hamster ovary cells were found to be unable to mediate I-apoJ internalization and degradation (data not shown). RAP, a competitor of apoJ binding to gp330 (Fig. 5), and unlabeled apoJ both acted to inhibit the F9 cell-mediated uptake and degradation of radiolabeled apoJ in a dose-dependent manner (Fig. 7). Lactoferrin, a gp330 ligand (Willnow et al., 1992) that was a poor competitor of gp330 binding to apoJ (), did not inhibit the degradation of apoJ (Fig. 7, C and D). The results show that cellular catabolism of apoJ correlates with the expression of gp330.


Figure 6: Internalization of I-gp330 monoclonal antibody by F9 cells. F9 cells were cultured in the presence or absence of RA and DBC for 7 days. Duplicate wells each containing 3 10 cells (RA- and DBC-treated or untreated) were incubated with I-gp330 monoclonal antibody 1H2 IgG (1.2 nM) for 4 h at 37 °C, 5% CO in the presence or absence of 200 molar excess unlabeled 1H2 IgG. The values indicated represent the amount of antibody specifically internalized as determined by subtracting the amount of I-1H2 IgG internalized in the presence of a 200 molar excess unlabeled 1H2 IgG from the amount of I-1H2 IgG internalized minus any unlabeled 1H2 IgG. These data are representative of five experiments, each performed in triplicate. Shown in the inset are immunoblotting analyses of detergent extracts from F9 cells (lane 1) and F9 cells treated for 7 days with RA/DBC (lane 2), which are stained with monoclonal anti-gp330 1H2.




Figure 7: Internalization and degradation of I-apoJ by F9 cells. Wells containing 3 10 F9 cells that had been either RA and DBC treated (panels B and D) or untreated (panels A and C) as described under ``Materials and Methods'' were incubated with I-apoJ (5 nM) for 18 h at 37 °C in the presence of increasing concentrations (1.8-450 nM) of unlabeled apoJ (), RAP (), or lactoferrin (). PanelsA and B show I-apoJ internalization data, and panels C and D show I-apoJ degradation data.



To provide further evidence for the role of gp330 as a mediator of apoJ endocytosis and degradation, we tested the effects of gp330 polyclonal antibodies (r239) on F9-mediated catabolism of I-apoJ. These antibodies have been shown to efficiently block the uptake and degradation of other gp330 ligands (data not shown). As shown in Fig. 8, both internalization (panel A) and degradation (panel B) of I-apoJ by RA/DBC-treated F9 cells were inhibited by gp330 antibodies, whereas antibodies to LRP had no effect. In addition, the degradation of I-apoJ was inhibited by chloroquine (Fig. 8, panel B), a drug that alters the pH of lysosomes leading to inhibition of lysosomal proteinase activity. These results suggest that gp330 functions to mediate endocytosis of apoJ, which leads to its degradation by lysosomal proteases.


Figure 8: Antibodies to gp330 inhibit internalization and degradation of I-apoJ by F9 cells. F9 cells treated for 7 days with RA/DBC were incubated with I-apoJ (5 nM) for 18 h at 37 °C in the presence of RAP (450 nM), polyclonal gp330 antibodies (100 mg/ml), polyclonal LRP antibodies (100 mg/ml), or chloroquine (100 mM). PanelA shows I-apoJ internalization data, and panelB shows I-apoJ degradation data. The observed effect of chloroquine on the level of apoJ internalization (panel A) is consistent with accumulation of the ligand within lysosomes.




DISCUSSION

While we have identified gp330 as a receptor capable of mediating apoJ endocytosis and degradation, we do not yet know the biological significance for such a process. ApoJ has been reported to bind to several proteins including the A4 peptide of the Alzheimer's precursor protein (Ghiso et al., 1993), a subclass of high density lipoprotein (de Silva et al., 1990a, 1990b; James et al., 1991; Stuart et al., 1992) and the membrane-attack complex C5b-C9 (Murphy et al., 1989; Jenne and Tschopp, 1989). It is conceivable that gp330 mediates clearance of apoJ in complex with these or other molecules. In this regard, apoJ would function like several other ligands for the LDLR family. For example, apoJ may function to target lipoproteins for clearance as does apoE or lipoprotein lipase (Kowal et al., 1989; Beisiegel et al., 1991; Chappell et al., 1992). ApoJ has also been shown to act as an inhibitor of the cytolytic activity of the complement membrane-attack complex C5b-C9 (Jenne and Tschopp, 1989; Kirszbaum et al., 1989). This interaction may facilitate clearance of the inactive complexes via gp330 in a manner analogous to the way in which proteases are inactivated by -macroglobulin or plasminogen activator inhibitor-1 and then cleared by LRP (Strickland et al., 1994).

A particularly important feature of apoJ is that its expression is up-regulated at sites undergoing tissue remodeling occurring in conjunction with apoptosis or following injury (Jenne and Tschopp, 1992). Increased levels of apoJ transcripts and/or protein have been measured in rat hippocampus following experimental injury (May et al., 1990), in prostate epithelial cells that atrophy following castration (Buttyan et al., 1989), in myocardiocytes adjacent to ischemic or inflammatory lesions of the heart (Vakeva et al., 1993),() in the regressing interdigital regions of the developing limb (Buttyan et al., 1989) and in retinitis pigmentosa-affected retinas (Jones et al., 1992; Jomary et al., 1993). The function that apoJ serves in these situations is not yet known. French et al.(1992) have proposed that apoJ may be involved in the removal of debris resulting from apoptosis or that it may protect by-stander cells from cytolysis by cell debris-activated complement. They also point out that in the thymus, where extensive apoptotic cell death normally occurs, inflammation is absent perhaps owing to the action of apoJ expressed by nondying cells in the medulla of the tissue. The typical absence of inflammation in areas that undergo apoptosis and the observed enhanced expression of apoJ in such areas makes it tempting to speculate that a primary function of apoJ is to restrict inflammation. Consistent with any of the postulated roles of apoJ is a role for a receptor such as gp330 to endocytose apoJ in complex with complement components or lipid debris/apoptotic bodies.

ApoJ has been found to be widely expressed by cells of reproductive, endocrine, and nervous tissues (Sylvester et al., 1991; Aronow et al., 1993; Sensibar et al., 1993; French et al., 1993; Laslop et al., 1993). For example, apoJ has been detected in ovary, prostate, seminal vesicle, testis, epididymis, kidney, liver, eye, and brain. ApoJ has also been detected in many body fluids including urine, saliva, seminal plasma, cerebral spinal fluid, and plasma (Blaschuk et al., 1983; Watts et al., 1990). There is a striking correlation between sites of apoJ expression and known sites of gp330 expression. Prominent expression of gp330 has been observed in the ependyma, choroid plexus, ciliary, and pigmented retinal epithelium, lung, kidney, yolk sac epithelium, and tissues of the male and female reproductive systems (Zheng et al., 1994; Kounnas et al., 1994). The colocalization of apoJ and gp330 strengthens the possibility that the two proteins interact with one another in vivo.

  
Table: Summary of binding constants for gp330-ligand interactions derived from homologous and heterologous ligand displacement experiments

The average equilibrium dissociation constants (K ) and average inhibition constants (K ) were derived from the best fit of the data to a single class of sites by using the computer program LIGAND (Munson and Rodbard, 1980). The K shown for gp330 binding to uPA:PAI-1 is in agreement with that reported by Moestrup et al. (1993b).



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK45598 (to W. S. A.) and GM42581 (to D. K. S.) and the Program of Excellence in Molecular Biology of the Heart and Lung HL41496 (to J. A. K. H.). 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.

§
Recipient of Individual National Research Service Award HL08744 from NHLBI, National Institutes of Health.

Recipient of a Merit Award from NHLBI, National Institutes of Health.

**
To whom correspondence should be addressed. Tel.: 301-738-0725; Fax: 301-738-0794; E-mail: argraves@hlsun.red-cross.org.

The abbreviations used are: gp330, glycoprotein 330; LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; apoJ, apolipoprotein J; apoE, apolipoprotein E; uPA, two chain urokinase; pro-uPA, prourokinase; PAI-1, plasminogen activator inhibitor-1; DBC, dibutyryl cAMP; TBS, Tris-buffered saline; tPA, tissue-type plasminogen activator; ELISA, enzyme-linked immunosorbent assay; RA, retinoic acid; PAGE, polyacrylamide gel electrophoresis.

Apolipoprotein J is synonymous with clusterin, sulfated glycoprotein-2 (SPG-2), complement lysis inhibitor (CLI), testosterone-repressed prostate message-2 (TRPM-2), Gp80 and SP-40-40.

S. Stefansson, D. A. Chappell, K. M. Argraves, and W. S. Argraves, manuscript in preparation.

D. Swertfeger, D. Witte, and J. A. K. Harmony, manuscript in preparation.


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