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Mar 19, 1990 - Communicated by B.Dobberstein. The low density lipoprotein receptor-related protein ... conversion of the N-linked carbohydrates to the mature.
The EMBO Journal vol.9 no.6 pp. 1 769 - 1776, 1990

Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in trans-Golgi compartment Joachim Herz, Robert C.Kowal, Joseph L.Goldstein and Michael S.Brown Departments of Molecular Genetics and Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA

Communicated by B.Dobberstein

The low density lipoprotein receptor-related protein (LRP) is a cell surface glycoprotein that binds and transports plasma lipoproteins enriched in apolipoprotein E. It is synthesized in the endoplasmic reticulum as a transmembrane glycosylated precursor that migrates with an apparent molecular mass of about 600 kd on SDS- polyacrylamide gels. After it reaches the Golgi complex, the protein is cleaved to generate two subunits with apparent molecular masses of -515 and 85 kd respectively. The larger NH2-terminal a-subunit lacks a membrane-spanning region. It remains attached to the membrane through noncovalent association with the smaller COOH-terminal $-subunit. Proteolysis occurs at the sequence RHRR, which resembles the sequence RKRR at the proteolytic site in the receptors for insulin and insulin-like growth factor-i (IGF-1), the only other cell surface receptors known to undergo proteolytic processing. Proteolysis of LRP occurs coincident with the conversion of the N-linked carbohydrates to the mature

endoglycosidase H-resistant, neuraminidase-sensitive form. Proteolysis is prevented by brefeldin A, which blocks transport to the Golgi complex. These data raise the possibility that LRP and the receptors for insulin and IGF-1 are processed by a specific endoprotease that recognizes protein with extended basic sequences and resides in the trans-Golg complex or in post-Golgi vesicles of the constitutive secretory pathway. Key words: LRP/trans-Golgi complex/trans-Golgi endoprotease/LDL receptor/secretory pathway Introduction The low density lipoprotein receptor-related protein (LRP), the largest cell surface protein characterized to date, is located on surface and intracellular membranes of many animal cell types (Herz et al., 1988; Kowal et al., 1989; Lund et al., 1989). This 600 kd glycoprotein binds plasma lipoproteins that have been enriched in vitro with excess amounts of apolipoprotein E (Kowal et al., 1989 and 1990; Beisiegel et al., 1989). It has been hypothesized to function as a receptor that clears chylomicron remnants and certain forms of very low density lipoproteins (VLDL) from the circulation. The NH2-terminal external domain of LRP comprises 4400 of the 4525 amino acids of the mature protein (Herz et al., 1988). It contains two types of 40 residue, cysteineOxford University Press

a

rich repeats, both of which are also found in the low density lipoprotein (LDL) receptor (Sudhof et al., 1985). By analogy with the LDL receptor, one class of repeats, shared with certain proteins of the terminal complement cascade, is believed to contain the ligand binding site (Esser et al., 1988). The other class, the so-called 'growth factor' repeat, is found in the extracellular domains of more than 20 proteins (Doolittle, 1985; Davis et al., 1987). The external domain of LRP is followed by a transmembrane region of 25 amino acids and a cytoplasmic tail of 100 amino acids. The LRP has been purified to homogeneity from rat liver and antibodies have been prepared (Kowal et al., 1989). The LRP on the surface of human fibroblasts was shown to bind apo E-enriched lipoproteins and to carry their cholesteryl esters into lysosomes where the cholesterol esters are hydrolyzed in a reaction that is inhibited by the lysosomotropic agent chloroquine. The released cholesterol is re-esterified in the cytoplasm (Kowal et al., 1989). Although this LRP-dependent uptake is likely to occur through receptor-mediated endocytosis, the cell surface binding and internalization events have not been demonstrated directly. The cytoplasmic domain of LRP contains two copies of the sequence, NPXY, which has been shown in the LDL receptor to be the targeting signal that mediates entry into coated pits (Chen et al., 1990). In the original study of LRP, an anti-peptide antibody directed against the COOH-terminal sequence was noted to react with a prominent lower molecular weight protein in the range of 85 kd (Herz et al., 1988). A similar 85 kd protein was seen when rat liver LRP was purified on a monoclonal antibody affinity column (Kowal et al., 1989) and when purified rat liver endosomes were immunoblotted with the anti-COOH terminal peptide antibody (Lund et al., 1989). It was hypothesized that this 85 kd protein represented a proteolytic breakdown product of the 600 kd LRP, but it has not been established whether this 85 kd fragment is produced physiologically in cells, or whether it is created artifactually after cell disruption. The current studies were designed to study the biosynthesis of LRP so as to determine the specific site at which the 85 kd fragment is produced. The data reveal that the 600 kd LRP undergoes physiological cleavage to produce a 515 kd protein and an 85 kd protein that remain noncovalently associated to form a functional 600 kd molecule.

Results Figure 1 shows a model for the structure of LRP that serves as the basis for the experiments in the current paper. We designate the intact protein as LRP-600 because it migrates at an approximate molecular weight of 600 000 on SDS-polyacrylamide gels. The external domain of LRP consists of alternating copies of ligand binding repeats (complement type repeats) and EGF precursor homology 1769

J.Herz et al.

LDL ReceptorRelated Protein

Ligand Binding Repeat

°-

Growth Factor Repeats

4- Spacer Region

EGF Precursor HHomology Domain

0 0-linked Sugar Domain

I Transmembrane Domain o3 EGF Repeat

Fig. 2. Biosynthesis and processing of 35S-labeled LRP in NRK cells. NRK cells were pulse-labeled for 1 h with [35S]cysteine as described

LRP-600

in Materials and methods. The cells were then chased with unlabeled

cysteine for the indicated time, after which detergent-solubilized cell extracts were immunoprecipitated with polyclonal anti-COOH terminal LRP antiserum. The immunoprecipitates were divided into three equal parts that were either not treated (nt) or treated with endoglycosidase H (EH) or neuraminidase (N) as described in Materials and methods. The samples were subjected to SDS-PAGE as described in Materials and methods. The gel was exposed to XAR-1 film (Kodak) for 48 h at -70°C. The positions of the uncleaved form of LRP (LRP-600) and of the cleaved form (LRP-515) are indicated.

LDL Receptor NH2

(SCOOH

SITE OF PROTEOLYTIC PROCESSING IN LRP 3914 3925 3935 HumanLRP

Rat LRP-85

APPTTSNRHRRQIDRGVTHLNI NH2 -Q ID RGVTNLSI

Fig. 1. Proteolytic processing of LRP. Upper Panel. Comparison of the structure of LRP and the LDL receptor. Brackets at the right denote the intact protein (LRP-600) and its two proteolytic components, LRP-515 and LRP-85. Lower Panel. Site of proteolytic processing in LRP. Purified rat LRP (- 100 pmol.) was subjected to 7.5% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride paper (Millipore) (Matsudaira, 1987). The band corresponding to LRP-85 was visualized with Coomassie Blue R-250 and subjected to NH2-terminal peptide sequence analysis on an Applied Biosystems 470A Protein Sequencer. The sequence obtained is displayed under the corresponding region of the predicted human LRP sequence (Herz et al., 1988). Question marks denote amino acid residues that were difficult to assign. Amino acid signals for each cycle were 5-6 pmol.

domains. The latter consist of cysteine-rich growth factor repeats (shown as open circles in Figure 1) that are separated by highly conserved spacer sequences of 280 amino acids (shown as wavy lines in Figure 1). The characteristic feature of each spacer segment is the occurrence of five copies of the YWTD motif at intervals of 50 amino acids. Homologs of all of these sequences also occur in the LDL receptor (Figure 1). To localize the site of proteolytic processing, we purified rat LRP by immunoaffinity chromatography as previously described (Kowal et al., 1989), subjected the protein to SDS -PAGE, and determined the amino acid sequence at the NH2-terminus of the 85 kd band. As shown in Figure 1 (lower panel), the rat sequence began with the glutamine that is found at amino acid position 3925 in the intact human protein. This glutamine is preceded by two consecutive arginines. This is a classic site for clipping of proteins that traverse the secretory pathway (Sossin et al., 1989). Cleavage at this site is predicted to give a large NH2-terminal fragment containing 3924 amino acids and a COOH-terminal fragment of 601 amino acids. Both of these -

-

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fragments migrate as somewhat larger species on SDS gels, owing in part to the presence of N-linked sugars (see below). Figure 2 shows a pulse-chase experiment designed to determine whether LRP was processed into the 515 kd form within the cell. For this purpose we used cultured rat kidney cells (NRK cells) which produce a relatively lar5ge amount of LRP. These cells were pulse-labeled with [3 S]cysteine

for 1 h and then chased with unlabeled cysteine for up to 4 h. Immediately after labeling (zero time in Figure 2), LRP was present as a 600 kd protein (lane 1). Treatment with endoglycosidase H (endo H) increased its mobility on reducing SDS -PAGE (lane 2), indicating that the N-linked carbohydrates were still in a high mannose, endo H-sensitive form. Consistent with this formulation, the protein was resistant to neuraminidase (lane 3). After a 1 h chase, more than half of the 600 kd form had been converted to a form that migrated slightly faster on SDS gels. We have designated this form LRP-515 (lane 4). This material was resistant to endo H (lane 5), but sensitive to neuraminidase (lane 6). By 2 h nearly all of the protein appeared as the 515 kd form (lane 7), which was resistant to endo H (lane 8) and sensitive to neuraminidase (lane 9). The same pattern persisted at 4 h (lanes 10-12). These data suggested that LRP was clipped to the 515 kd form at approximately the same time that the N-linked carbohydrates were processed to the endo Hresistant, neuraminidase-sensitive form, which implies that the clipping occurred after the protein was transported to a late Golgi compartment (Kornfeld and Komfeld, 1985). To further study the role of the Golgi complex in LRP processing, we employed brefeldin A, which blocks the transport of proteins and membrane vesicles from the endoplasmic reticulum to the Golgi complex (LippincottSchwartz et al., 1989). At zero time after the labeling period, LRP was present in the neuraminidase-resistant 600 kd form (Figure 3, lanes 1 and 2). Brefeldin A had no effect on the structure of this precursor (lane 4). At this time point the 85 kd fragment was undetectable. After 1 h, 50% of LRP had been processed to a 515 kd, neuraminidase-sensitive form (lanes 5 and 6). Simultaneously, the 85 kd fragment appeared and this was also neuraminidase-sensitive (lanes 5 and 6). In the presence of brefeldin A, LRP remained in the 600 kd, neuraminidase-resistant form, and no 85 kd fragment appeared (lanes 7 and 8). By 3 h in control cells, virtually 100% of the detectable LRP had been converted to the 515 kd and 85 kd, neuraminidase-sensitive forms, but -

Proteolytic processing of LRP

Fig. 3. Inhibition of proteolytic processing of LRP-600 by brefeldin A. NRK cells were pulse-labeled for 1 h with [35S]cysteine and chased with unlabeled cysteine for the indicated time in the presence or absence of 5 ug/ml of brefeldin A. In one group of cells (lanes 13- 18), after a 3 h chase the medium was replaced with DMEM without brefeldin A, and the drug was allowed to wash out of the cells for the indicated time (washout period). Detergent-solubilized cell extracts were immunoprecipitated with polyclonal anti-COOH terminal LRP antiserum. The immunoprecipitates were divided into two equal parts that were treated with or without neuraminidase as described in Materials and methods. The samples were subjected to SDS-PAGE, after which the gel was exposed to XAR-1 film for either 2.5 h (LRP-600/515) or 16 h (LRP-85). The positions of the various forms of LRP (LRP-600/515 and 85) are indicated. The decreased intensity of LRP 600/515 in lane 3 is due to an artifact in the gel.

neither of these forms appeared in the presence of brefeldin A (lanes 9-12). At this point, we washed out the brefeldin A. Within 1 h, the neuraminidase-sensitive, 515 kd and 85 kd forms appeared (lanes 15 and 16). By 3 h, all of the previously synthesized protein was in the neuraminidasesensitive, 515 and 85 kd forms (lanes 17 and 18). These data confirm the suggestion that proteolytic processing of the LRP does not occur until after it is transported to a late compartment of the Golgi complex. The 515 kd and 85 kd forms of metabolically labeled and immunoprecipitable forms of LRP do not appear to be covalently associated since they can be completely separated on nonreducing SDS -PAGE (data not shown). Upon reduction, LRP-5 15, like the LDL receptor (Daniel et al., 1983), shows a markedly slower mobility on SDS gels, owing to disruption of the extensive disulfide bonding of the ligand binding repeats (data not shown). Our previous observation that the 85 and 515 kd forms of rat liver LRP co-purify on affinity chromatography with an anti-COOH terminal antibody (Kowal et al., 1989) raised the possibility that the two fragments remain associated with each other after proteolytic cleavage. To test this hypothesis further, we subjected LRP to a series of protein separation steps and then determined whether the two fragments had been separated. Figure 4 shows an experiment in which a crude extract of rat liver membranes was applied to a DEAE -cellulose column and eluted with a salt gradient. Fractions were subjected to nonreducing SDS -PAGE and assayed for the presence of the 600 kd and 85 kd fragments by immunoblotting with a monoclonal anti-COOH-terminal antibody (Figure 4A) and for the 600, 515 and 85 kd forms by incubation with a polyclonal antibody directed against the entire protein (Figure 4B). The 515 and 85 kd fragments eluted together in fractions 15-27 of this column. The pooled DEAE-cellulose fractions containing LRP were applied to a column that contained a covalentlybound monoclonal antibody directed against the COOHterminal tail of LRP. Fractions were eluted, subjected to SDS -PAGE, and tested for all three forms of LRP by probing with the affinity-purified polyclonal anti-LRP antibody (Figure SA) or for the 600 kd and 85 kd forms by probing with the monoclonal anti COOH-terminal IgG 1 1H4 (Figure SB). The starting material contained both the

0

-- .X 4....

Fig. 4. Co-elution of hepatic LRP-515 and LRP-85 on DEAE-cellulose chromatography. Triton X-100-solubilized membranes from 25 g of rat liver (Kowal et al., 1989) were applied to a 50 ml DEAE-cellulose column (2.6x9.5 cm). The column was washed with 150 ml of buffer as described (Kowal et al., 1989), eluted with 150 ml of 0-1 M NaCI linear gradient, and collected in 4 ml fractions. Samples (20 1I) from alternating fractions were subjected to nonreducing 6% SDS-PAGE on a Mini-Protean II apparatus (Biorad) at 150 V for 45 min at room temperature. Protein was transferred to nitrocellulose paper for 1 h at 1 A and subjected to immunoblot analysis with either 5 pig/ml of monoclonal anti-LRP IgG-l 1H4 (Panel A) or 2 ug/ml of polyclonal anti-LRP IgG (Panel B), followed by incubation with 125I-labeled rabbit anti-mouse IgG (Panel A) or 125I-labeled goat anti-rabbit IgG (Panel B), each added at 180 ng/ml with a specific activity of -4000 c.p.m./ng. The blots were exposed to XRP-1 film from 12 h at room temperature with an intensifying screen.

515 and 85 kd forms (lane S). A small amount of both forms appeared in the flow-through (lane 1), but none was detected in the washes (lanes 2-4). When eluted with ammonium hydroxide, the 515 kd LRP and the 85 kd LRP appeared together in fractions 8 and 9. The 515 kd component did not react with the anti-COOH terminal antibody (Figure SB) even after a prolonged exposure (not shown), confirming that the large fragment in Figure SA was the 515 kd NH2-terminal portion and excluding the possibility that it

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Fig. 5. Co-retention of LRP-515 and LRP-85 on affinity chromatography with monoclonal anti-COOH terminal antibody. Pooled rat hepatic DEAE-cellulose fractions dialyzed against Buffer B (106 mg in 100 ml) were subjected to affinity chromatography on a column containing monoclonal anti-LRP IgG-11H4 as described in Materials and methods. Samples (50 i1 each) of the starting material (lane S), the material that flowed through the column (lane 1), the fractions from the Buffer C wash (lanes 2 and 3), the NH4HCO3 wash (lane 4) and samples (5 /.l each) of fractions eluted with NH40H (lanes 5-12) were subjected to nonreducing 3-8% SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was performed with affinity-purified polyclonal anti-LRP IgG (Panel A) or 2 pg/mi of monoclonal anti-LRP IgG-l lH4 (Panel B) as described in the legend to Figure 4. The blots were exposed with intensifying screens to either XRP-1 film for 14 h at -70°C (Panel A) or XAR-1 film for 7 h at -70°C (Panel B).

Fig. 7. Immunoblot analysis of rat liver LRP after crosslinking with glutaraldehyde. Pooled rat hepatic DEAE-cellulose fractions dialyzed against Buffer B (-2.5 mg protein) were concentrated on a 1.5 ml DEAE-cellulose column that had been equilibrated with Buffer B containing 1% (v/v) Triton X-100. The column was washed with 20 ml of Buffer E (25 mM sodium HEPES, 50 mM NaCl, 2 mM CaC12, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride at pH 7.5). Protein was eluted with 1 M NaCl in Buffer E and dialyzed against 3 1 of Buffer E containing 0.1 M NaCl. Aliquots of 10 fig protein in 5 IJ were diluted to a final volume of 75 1l with Buffer E containing 0.5 M sodium thiocyanate. The samples were treated for 45 min at room temperature with 2.5 ll of distilled water containing glutaraldehyde to achieve the indicated final concentration. The treated samples were subjected to nonreducing 3-8% SDS-PAGE and immunoblot analysis with anti-LRP monoclonal IgG-l lH4 as described in the legend to Figure 4. The blots were exposed to XAR-1 film for 8 h at -70°C with an intensifying screen.

represented a small amount of intact 600 kd LRP that was retained on the column. The immunoaffinity column gave some enrichment for the 85 kd component relative to the 515 kd component. This enrichment is likely to be due to elution of some of the 515 kd component during the extensive

washing.

Fig. 6. Co-elution of LRP-515 and LRP-85 on Superose-6 FPLC. Pooled rat hepatic DEAE-cellulose fractions dialyzed against Buffer B (-4 mg protein) were concentrated on a 1.5 ml DEAE-cellulose column that had been equilibrated with Buffer B containing 1 % (v/v) Triton X-100. The column was washed with 15 ml of Buffer D (Buffer B containing 40 mM octylglucoside). Protein was eluted with 1 M NaCl in Buffer D and dialyzed against 50 ml of Buffer D containing 0.1 M NaCl for 12 h at 4°C. An aliquot (0.9 mg protein) was subjected to gel-filtration on a 25 ml Superose 6 column in Buffer D with 0.1 M NaCl and collected in 0.5 ml fractions. Samples (50 Al each) of the void fraction (lane V) and subsequent alternating fractions were subjected to nonreducing 3-8% SDS-PAGE and stained with silver (Panel D) or transferred to nitrocellulose and subjected to immunoblot analysis with either affinity-purified polyclonal anti-LRP IgG (Panel B) or 2 Ag/mni of COOH-terminal specific monoclonal antiLRP IgG-l lH4 (Panel C) as described in the legend to Figure 4. The blots were exposed with intensifying screens to either XRP-1 film for 24 h at -70°C (Panel B) or XAR-1 film for 2 h at -70°C (Panel C). Panel A shows the migration of the following molecular weight markers on Superose 6 under identical conditions: thyroglobulin, 670 kd; ferritin, 440 kd; catalase, 232 kd; gamma globulin, 158 kd; ovalbumin, 44 kd.

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To further confirm that the 85 kd fragment remained in association with the 515 kd fragment, we subjected the DEAE cellulose-purified LRP to gel filtration on a Superose column (Figure 6). The 85 kd fragment emerged from this column together with the 515 kd fragment in advance of the 670 kd thyroglobulin marker protein, indicating that it was part of a large complex. An association of the 85 kd fragment with the 515 kd fragment was also suggested by a chemical crosslinking experiment (Figure 7). DEAE cellulose-purified rat liver LRP was incubated with increasing concentrations of glutaraldehyde, subjected to SDS-PAGE and immunoblotted with the monoclonal anti COOH-terminal antibody. At glutaraldehyde concentrations > 0.05 mM, the 85 kd fragment was crosslinked to a form that migrated with an apparent molecular weight corresponding to the 600 kd form (Figure 7). This crosslinking occurred in the presence of 0.5 M sodium thiocyanate, a chaotropic agent that dissociates proteins that are joined by weak ionic interactions, suggesting that the association of the 85 and 515 kd forms is stable. To assess the stoichiometry of the complex between the 515 and 85 kd forms, we labeled NRK cells with [35S]cysteine or [35S]methionine, immunoprecipitated LRP with an anti-COOH-terminal antibody, subjected the protein to SDS -PAGE and determined the amount of radioactivity in the 515 and 85 kd bands with a quantitative radioanalytic imaging system. The experiment was performed in triplicate (Table I). We used the known amino acid composition of human LRP to calculate the ratio of radioactivity to be expected in the 515 and 85 kd bands if these fragments were

Proteolytic processing of LRP Table I. Relative amounts of steady state levels of LRP-515 and LRP-85 in NRK cells Sample

Isotopic precursor

35S-radioactivity in: LRP-515

LRP-85

(a)

(b)

Ratio (a/b) Observed Expected

counts/band Exp. A 1 2 3 Exp. B. 4 5 6

[35S]cysteine

21 755 37 040 18 791

3302 5678 2162

6.6 6.5 8.7

6.9 6.9 6.9

[35S]methionine 5476 [35S]methionine 10 062 [35S]methionine 5278

1243 2013 2104

4.4 5.0 2.5

4.1 4.1 4.1

[35S]cysteine [35S]cysteine

Monolayers of NRK cells were grown as described in Materials and methods. On day 5, replicate 60 mm dishes (samples 1-3) were each pulselabeled for 2 h with 66 itCi/ml [35Slcysteine in cysteine-free DMEM followed by a 7 h chase in DMEM containing 0.2 mM unlabeled cysteine. Three other replicate dishes (samples 4-6) were radiolabeled in the same way except that 66 PCi/ml [35S]methionine was used as the isotopic precursor in methionine-free DMEM, and the chase medium contained 0.2 mM unlabeled methionine. At the end of the chase, the medium was removed, monolayers were washed, and detergent-solubiized extracts were immunoprecipitated with polyclonal anti-COOH terminal LRP antiserum and subjected to electrophoresis on SDS -PAGE as described in Materials and methods. Radiolabeled bands corresponding to LRP-515 and LRP-85 were quantified by scanning the gel for 12 h on an AMBIS Radioanalytic Imaging System using a 1.6 mm circular resolution plate. Background radioactivity (- 1700 counts for each sample) was measured in a representative area of each lane and was subtracted from the total counts to give the values shown. The expected ratio of 35S-radioactivity in rat LRP-515 and LRP-85 was calculated from the number of cysteine and methionine residues encoded in the cDNA sequence of human LRP (Herz et al., 1988).

present in a 1:1 molar ratio. For both labeled amino acids, the observed ratios were quite close to the predicted ones, indicating the presence of equimolar concentrations of the 85 and 515 kd fragments in mature LRP. To determine the intracellular distribution of LRP, we fixed NRK cells in situ, permeabilized them with Triton X-100, and then incubated them with an affinity-purified polyclonal anti-LRP antibody (Figure 8, panel 1) or a polyclonal anti-COOH-terminal LRP antibody (panel 2). For comparison, we also stained the cells with a polyclonal antiLDL receptor antibody (panel 3). Both anti-LRP antibodies gave the same receptor distribution. They showed the receptor in peripheral vesicles and as a closely-packed cluster of amorphous fluorescent dots in a distribution corresponding to the Golgi complex. This intracellular distribution was indistinguishable from that of the LDL receptor in the same cells.

Discussion The current results demonstrate that the 4525 amino acid LRP is synthesized as a glycosylated precursor that migrates with an apparent molecular weight of about 600 000 on SDS -PAGE. After it reaches the Golgi complex, it is proteolytically clipped to produce two components of 3924 and 601 residues with apparent molecular masses on SDS gels of 515 and 85 kd respectively. The 85 kd COOHterminal component undergoes no further proteolytic cleavage since its NH2-terminal sequence remains intact (Figure 1) and it continues to react with an anti-peptide antibody directed at the extreme COOH-terminus. We cannot

exclude the possibility that the 3924 residue NH2-terminal component might undergo some additional cleavage at its COOH-terminus, but extensive trimming is unlikely as judged from the apparent size of the component on SDS-PAGE. The proteolytic processing of LRP occurs at about the same time that the N-linked carbohydrates are converted to the endoglycosidase H-resistant form and the terminal sialic acids are added. We never observed a neuraminidaseresistant or endoglycosidase H-sensitive form of the 85 kd subunit. Proteolysis is prevented by treatment of the cells with brefeldin A, which blocks transport to the Golgi complex (Figure 3). It is also blocked by monensin (data not shown), which inhibits transport from the cis to transGolgi (Tartakoff, 1983). Together, these findings suggest that the processing of LRP occurs in the terminal cisternae of the trans-Golgi complex or in a secretory vesicle that is en route from the Golgi complex to the cell surface. Although proteolytic processing of viral membrane glycoproteins and secretory proteins is common (Tartakoff, 1983; Sossin et al., 1989), cleavage of cell surface receptors is distinctly unusual. The only three well-characterized examples involve the closely related receptors for insulin and insulin-like growth factor-I (IGF-I) (Hedo et al., 1983; Olson et al., 1988; Ullrich et al., 1985 and 1986) and the polymeric IgA receptor (Mostov et al., 1984). It is striking that the amino acid sequence at the site of proteolysis of LRP (RHRR) resembles the extended basic sequence at the cleavage site for the insulin and IGF-I receptors (RKRR), which suggests that all three proteins may be clipped by a specific endoprotease that recognizes an extended basic sequence and resides in the Golgi complex or in post-Golgi vesicles of the constitutive secretory pathway. In contrast, the polymeric IgA receptor, which after cleavage releases the majority of its extracellular domain termed 'the secretory component', does not display such a tetrabasic sequence at its cleavage site (Mostov, et al., 1984; Eiffert, et al., 1984). The tetrabasic site of proteolytic clipping in LRP occurs within the eighth EGF precursor homology region, which is the one that is closest to the plasma membrane (Figure 1). This is the only EGF precursor homology region that contains the tetrabasic RHRR sequence for proteolytic clipping. This sequence does not occur in the LDL receptor, nor does it occur in the EGF precursor itself. The sequence site for proteolytic clipping must be conserved in evolution since proteolytic clipping has been demonstrated for the human, rabbit, rat and mouse LRP (Herz et al., 1988; Kowal et al., 1989; and unpublished observations). After the clipping occurs, the 515 and 85 kd components of LRP remain associated in a 1: 1 stoichiometric ratio. The attachment is noncovalent since the fragments can be separated on SDS -PAGE in the absence of sulfhydrylreducing agents. The complex is extremely tight since the two proteins co-purify on ion exchange and gel filtration columns and since they can be crosslinked to each other by chemical crosslinking agents even in the presence of 0.5 M sodium thiocyanate. This finding suggests that there is some complementarity between a sequence in the 515 kd component and a sequence in the 85 kd component. This is likely to represent an interaction between one or more of the complement-type repeats or growth factor repeats in the 515 kd component with one or more of the EGF-type repeats in the 85 kd component. Since the 515 kd and 85 kd

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Fig. 8. Intracellular localization of LRP in NRK cells by immunofluorescence; comparison with LDL receptors. Monolayers of NRK cells were seeded on day 0 in 6-well dishes containing sterile coverslips at a density of 3 x 104 cells/well in DMEM supplemented with penicillin/streptomycin and 10% fetal calf serum. On day 2, the medium was switched to DMEM containing 10% lipoprotein-deficient newborn calf serum (Goldstein et al., 1983). On day 4 the medium was removed and the wells were washed twice with PBS. Cells were fixed for 15 min at 22°C in 3% (w/v) paraformaldehyde dissolved in PBS and washed once with PBS. Residual paraformaldehyde was quenched by adding 50 mM NH4Cl dissolved in PBS for 30 min at 4°C. The coverslips with the fixed monolayers were then treated with 0.2% Triton X-100 in PBS for 5 min followed by 30 min at 22°C with 5% bovine serum albumin in PBS (blocking buffer). The indicated primary antibodies (see below) were added to the wells in a total volume of 750 yl and incubated for 1 h at 22°C. The coverslips were washed 5 times with PBS (5 min/wash) and then incubated for 30 min at 22°C in the dark with 3 yig/ml of FITC-conjugated goat anti-rabbit IgG (Zymed Laboratories, Inc.) dissolved in blocking buffer. The PBS washes were repeated, and the coverslips were rinsed once in distilled water just prior to mounting. Photographs of the fluorescent images were taken on a Zeiss RSIII fluorescence microscope. The primary antibodies used were as follows: affinity-purified polyclonal anti-LRP IgG (Panel 1); a 1:250 dilution of polyclonal anti-COOH terminal LRP antiserum (Panel 2); 5 Ag/ml of polyclonal anti-LDL receptor (Panel 3); and 5 tig/ml of nonimmune rabbit IgG (Panel 4). Magnification, 117 x.

components appear to form subunits of a 600 kd holo-LRP, we suggest that these components be designated a- and ,subunits, respectively. At present we do not know whether the functional form of the cleaved LRP is an a,B monomer, and a232 dimer, or some higher order multimer. In several experiments we noted that a small and variable percentage of [35S]cysteine labeled LRP remains in a 600 kd form even after a prolonged chase of several hours with unlabeled cysteine. This 600 kd LRP does not appear to reach the cell surface. It remains neuraminidase-resistant and endoglycosidase H-sensitive. Moreover, unlike the 515/85 kd complex, the 600 kd LRP is not degraded when intact NRK cells are incubated for 5 h with an antibody

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against LRP (Kowal et al., 1990; and unpublished observations). We have not been able to purify enough LRP-600 to determine whether or not it binds apo E-enriched lipoproteins. The functional reason for the proteolytic clipping of LRP remains obscure. In principle, this clipping might be necessary for the formation of a receptor that is competent to bind apo E-enriched lipoproteins. It might also permit subunit exchange of the 515 kd component among receptors or lead to the release of the 515 kd component from the 85 kd component, either on the cell surface or in an intracellular vesicle during recycling. However, we were never able to detect any LRP-515 in culture medium of metabolically

Proteolytic processing of LRP

labeled cells (data not shown). Further studies will be necessary to determine whether the 515 kd subunit ever becomes separated from the 85 kd subunit during the functional lifetime of an LRP molecule. Most multimeric cell surface proteins are assembled by one of two mechanisms. The first type, exemplified by the receptors for insulin and IGF-I, arises from a single polypeptide chain that is cleaved proteolytically to form two subunits that are covalently attached by disulfide bonds to form ae-f-f-ae heterodimers (Olson et al., 1988). The second type, exemplified by MHC class I molecules, consists of two noncovalently bonded protein chains, each of which is the product of a different gene and neither of which undergoes proteolytic processing (Ploegh et al., 1981). The LRP is distinctly unusual in that it is synthesized on a single polypeptide chain that is cleaved into two subunits that remain attached to each other noncovalently without the necessity for disulfide bonds.

Materials and methods Reagents Neuraminidase (Type X from Clostrinliuottz perfringens) was obtained from Sigma Chemical Co. (Cat. No. N2133). Endoglycosidase H from Streptomrvces livicvdns was purchased from Boehringer-Mannheim (Cat. No. 100-119). Protein A-Sepharose beads. CNBr-activated Sepharose 4B. and Superose 6 FPLC (prepacked HR 10/30) columns were obtained from Pharmacia. Brefeldin A was purchased from Epicentre Technologies (Madison. WI). DEAE-cellulose (DE-52) was purchased from Whatman Biosystems. Ltd.

Antibodies against LRP Polyclonal anti-LRP IgG was prepared by immunizing rabbits with purified rat LRP and isolating IgG as described (Kowal et al., 1989). A polyclonal rabbit antiserum against a synthetic peptide corresponding to the COOHterminal 13 amino acids of human LRP was prepared as described (Herz et /l., 1988) and is designated as polyclonal anti-COOH terminal LRP antiserum. A mouse monoclonal antibody against a synthetic peptide corresponding to the COOH-terminal 13 amino acids of human LRP was prepared as described (Kowal et al., 1989) and is designated as monoclonal anti-LRP IgG-l H4. A polyclonal rabbit antibod) against purified bovine LDL receptors was prepared as described (Russell et a!., 1984) and is designated as polyclonal anti-LDL receptor IgG. Cell culture and immunoprecipitation Stock cultures of normal rat kidney cells (NRK cells. clone SA6; obtained from J.E.DeLarco. National Cancer Institute. NIH. Bethesda. MD) were grown in monolayer culture at 37°C in a 10%c CO, incubator. Cells were

set up for experiments according to a standard format. On day 0. trypsinized cells were seeded in 60 mm Petri dishes at a density of 3 x 104 cells/dish in Dulbeco's modified Eagle medium (DMEM) supplemented with 100 U/ml penicillin. 100 lig/ml streptomycin sulfate, and 10% (v/v) fetal calf serum. On day 2. the medium was replaced with fresh mediuni of the same composition. On day 4. the cells were washed once with phosphate-buffered saline (PBS) and switched to 1.5 ml of cvsteine-free DMEM containing penicillin. streptomycin. 25 mM HEPES (pH 7.2) and 10% fetal calf serum. After incubation for 20 min at 37°C. each monolayer received 66 1xCi/ml [35S]cysteine (New England Nuclear). After incubation for 1 h at 37°C (pulse). 1.5 ml of DMEM containing 0.2 mM unlabeled cysteine. 25 mM HEPES (pH 7.2). and 10%7 fetal calf serum wvere added to each dish. and the incubations were continued for the indicated time (chase). After the chase period, the medium was renmoved and the cells were lyzed in the dish with I ml of buffer containing 20 mM Tris-HCl. 150 mM NaCl. 2 mM MgCl,. 1 mM phenylmethylsulfonyl fluoride, and 1 %k (v/v) Nonidet P40 at pH 7.5. Nuclei were removed by centrifugation at 12 OOOg for 2 min in an Eppendorf microfuge. Each supermatant was transferred to a new tube and incubated on ice for 1 h with 10 1l of polyclonal anti-COOH terminal LRP antiserum. followed by addition of 70 ,il of a 1: 1 slurry of Protein A-Sepharose equilibrated in Buffer A (20 mM Tris-HCI. 150mM NaCI. 2 mM MgCI,. and 0.2%' Nonidet P-40 at pH 7.5). After incubation for 30 min at 4°C on a rotating wheel. the Protein A-Sepharose beads were sedimented by a brief spin (5 s) in an Eppendorf microfuge and the super-

natant was removed by suction. The beads were washed three times in Buffer A and once in Buffer A containing 0.5 M NaCl. After one additional wash with PBS. the beads were transferred to new tubes for incubation with or

without glycosidase (see below).

Glycosidase treatment and SDS -PAGE of immunoprecipitates An aliquot of the immunoprecipitate from one dish of 35S-labeled cell extract (see above) was incubated at 20°C for 16 h with 0.3 U neuraminidase in 30 1t of 20 mM sodium citrate, 2 mM CaCI2 and 150 mM NaCl at pH 6. Another aliquot of the same immunoprecipitate was incubated at 20°C for 16 h with 0.05 U endoglycosidase H in 30 A1 of 10 mM sodium phosphate at pH 7. For control incubations, an equivalent amount of immunoprecipitate was incubated in 30 ttl of the neuraminidase buffer without any enzyme. The reactions were stopped by the addition of an equal volume of 2 x SDS sample buffer (Laemmli, 1970) containing 5%c (v/v) A3-mercaptoethanol. Samples were boiled for 5 min and subjected to 3-8%7c SDS-PAGE (Kowal et al., 1990). Purification of LRP LRP was purified from rat livers as previously described (Kowal et al., 1989) with the following modifications. DEAE-cellulose fractions containing partially purified LRP were pooled and dialyzed against Buffer B (50 mM Tris-HCI. 50 mM NaCl. 2 mM CaCl,, and 1 mM phenv'lmethylsulfonyl fluoride at pH 7.5) for at least 12 h at 40C. The dialysate was applied to a column containing the COOH-terminal specific mouse monoclonal anti-LRP IgG- 11 H4 coupled to cyanogen bromideactivated Sepharose 4B. The column was washed with two 100 ml fractions of Buffer C (50 mM Tris-HCI. 2 mM CaC1,. 40 mM octylglucoside, and 1 nmM phenylmethylsulfonryl fluoride at pH 7.5), followed by 6 ml of 50 mM NH4HCOI. LRP was eluted with 0.1 M NH40H in ten I ml fractions, frozen in liquid N,, and lyophilized. For experiments, fractions of affinitypurified LRP were resuspended in 0.2 ml of 50 mM Tris-HCI and 2 mM CaCI, at pH 8.

Immunoblot analysis

Immunoblotting of LRP was performed as previously described (Kowal et /l., 1989) with either anti-LRP monoclonal IgG- 1 H4 or affinity-purified

polyclonal anti-LRP IgG (see below). The gels were calibrated with the following molecular weight markers: myosin. 200 kd; 3-galactosidase. 116 kd: phosphorylase B. 98 kd; bovine serum albumin. 66 kd.

Affinity-purified polyclonal anti-LRP IgG DEAE-cellulose fractions containing partially purified rat LRP were subjected to preparative nonreducing 3-8% SDS-PAGE and transferred to nitrocellulose paper as described (Kowal et al., 1989 and 1990). The blots were stained with Ponceau S (Sigma Chemical Co.) and the band corresponding to LRP-600/515 w\as cut out and incubated with polyclonal anti-LRP IgG (5 yig/ml) at 4°C for 16 h. The cut nitrocellulose strip was washed extensively with PBS, and bound antibody was eluted with 0.1 M glNcine-HCI and 0.2% (w/v( gelatin at pH 2.35 for 5 min at 4°C. The solution was neutralized by addition of 6 vol of 0.5 M Tris-HCI and 0.1% gelatin at pH 8.5 and then used for immunoblotting within 24 h. For

immunofluorescence.

concentration of 3%

bovine

(w'/v).

serum albumin was added to a final

Acknowledgements We thank Carolyn

R.Moomaw and Joan T.Hsu for help with amino acid

sequence analysis: Wen-Ling Niu for excellent technical assistance; and

Edith Womack for invaluable help with growing cultured cells. R.C.K. is a Medical Scientist Training Grant (GM 08014) from the National Institutes of Health. This work was supported by grants from the National Institutes of Health (HL 20948), the Lucille P.Markey Charitable Trust and the Perot Family Foundation.

supported by

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