INTRODUCTION

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The low density lipoprotein (LDL)¹ receptor-related protein (LRP), a member of the LDL receptor gene family, adopts the topology of a type I integral membrane protein (1). A major structural feature of this family of receptor proteins is the arrangement of clusters of ligand-binding repeats (designated class A repeats) interspersed between clusters of epidermal growth factor precursor repeats within the extracellular domains (2). Variations in the makeup of the modular extracellular domains appear to dictate the ligand binding characteristics of the various family members, which in the case of LRP shows multifunctionality for a wide range of proteins, including ₂-macroglobulin (₂M) and Pseudomonas exotoxin A (PEA). However, all of the members of this receptor family share a capacity to bind a 39-kDa receptor-associated protein, or RAP (3). Furthermore, the cytoplasmic domain of LRP contains two copies of the NPXY motif that is important for the clathrin-coated pit-mediated internalization of the LDL receptor (4).

LRP is translated as a single polypeptide (600 kDa) consisting of 4525 amino acids in humans (5) and 4522 amino acids in chickens (6). A distinct structural feature that sets LRP apart from the other LDL receptor family members is that LRP undergoes post-translational proteolytic processing (7). Processing occurs in the trans-Golgi compartment 1-2 h after LRP translation (7) and is presumably catalyzed by the endopeptidase furin (8), which recognizes the RXRR consensus sequence, RHRR in human LRP (5) and RNRR in chicken LRP (6). This post-translational processing results in the formation of mature LRP as a noncovalently associated heterodimer, consisting of the extracellular 515-kDa -chain and the transmembrane 85-kDa -chain (7). Proteolytic processing by furin also occurs in other membrane proteins (e.g. the insulin receptor), and sometimes a crucial function for processing becomes evident. For instance, a mutation at the consensus sequence of the human insulin receptor (i.e. RNRR-to-RNRS substitution) that results in defective processing leads to impaired receptor function and severe insulin resistance (9, 10).

The current knowledge of the biological significance of post-translational LRP processing is limited. In several studies, expression of membrane-anchored LRP minireceptors, which contained a portion of the extracellular domain plus the transmembrane and intracellular sequences but lacked the processing site, has yielded functional receptors. Willnow et al. (11) have expressed such a LRP minireceptor that bound and degraded the 39-kDa RAP and the tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex. Recently, we have expressed two membrane-anchored chicken LRP minireceptors and found that both of the unprocessed receptors restored the toxicity of PEA and degradation of ₂M in transfected LRP-null cells (12). However, the role of post-translational processing in the function of the full-length LRP has not been analyzed. In the current study, we created a full-length chicken LRP processing mutant in which Arg³⁹⁴² within the furin cleavage site (RNRR³⁹⁴²) (6) was replaced by Ser and examined the requirement for processing in the endocytic functions and intracellular trafficking of LRP.

	EXPERIMENTAL PROCEDURES

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MaterialsDNA restriction and modification enzymes, endoglycosidase H (Endo H), and peptide:N-glycosidase F were purchased from New England Biolabs. Neuraminidase was purchased from Boehringer Mannheim. ProMix^TM (a mix of [³⁵S]methionine and [³⁵S]cysteine; 1000 Ci/mmol), carrier-free Na¹²⁵I, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody, and the enhanced chemiluminescence (ECL) reagents for immunoblotting were obtained from Amersham Pharmacia Biotech. Bicinchoninic acid protein assay reagent and EZ-Link^TM NHS-SS-Biotin (sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate) were obtained from Pierce. Bacterially expressed recombinant PEA was prepared as described (13). The glutathione S-transferase/RAP expression plasmid containing the human RAP cDNA was obtained from D. Strickland (American Red Cross, Rockville, MD). The purification of glutathione S-transferase/RAP and subsequent thrombin cleavage of the fusion protein were carried out as described previously (14).

Preparation of Mutant LRP Expression Plasmid-- The parental expression plasmid pcLRP-wt that encoded the full-length chicken LRP (originally designated pcLRP-100) was constructed in the pCMV5 vector using cDNA fragments as described previously (12). A fragment that extended from nucleotide 9112 (an XhoI site) of the LRP cDNA to another XhoI site (in the pCMV5 vector) was subcloned into pBluescript SK⁺ (Stratagene) to create pLRP3' for mutagenesis purposes. To abolish cLRP proteolytic processing, the cDNA sequence encoding the furin cleavage site, RNRR³⁹⁴² (6), was altered to encode RNRS by polymerase chain reaction-based mutagenesis. Two mutagenic primers, cla1 (5'-GAGAACGTGCGCATCGATGCC-3') and sac2rs (5'-ACCCCGCGGCATCTTCAGCCCGGAGATGTTCAGGTGGGTGACGCCGCCGTCGATCTGCGATCGGTTGCGG-3'), were used to amplify the ClaI-SacII (nucleotides 12969-13148 of the chicken LRP cDNA) fragment that encompassed the RNRR³⁹⁴² coding sequence. The sac2rs primer changed the encoded RNRR into RNRS and also introduced a PvuI site (underlined). Amplification of the ClaI-SacII fragment was achieved by polymerase chain reaction using Vent_R^TM polymerase (New England Biolabs). The mutant ClaI-SacII polymerase chain reaction product was inserted into pLRP3' that had been digested with ClaI and SacII, and finally a XhoI-XhoI fragment of the mutant pLRP3' was inserted into pcLRP-wt that had been digested with XhoI, to create the full-length mutant plasmid pcLRP-RS (Fig. 1A). The nucleotide sequences of the resulting mutant constructs were verified by double-stranded DNA sequencing with T7 DNA polymerase (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Cell Culture and Transfection-- Wild type CHO-K1 and the LRP-null CHO cell line (15) were cultured in Ham's F-12 medium containing 10% fetal bovine serum as described previously (12). Stable transfection into LRP-null CHO cells (in 100-mm dishes) was achieved by cotransfection of 15 µg of pcLRP-wt or pcLRP-RS with 0.15 µg of pSV2neo (12). Stable transformants were selected using 700 µM G418 and maintained with 500 µM G418.

Northern Blot Analysis-- Total RNA was isolated from confluent cells (100-mm dish) using the TRI^TM reagent (Molecular Research Center) and resolved (20 µg) by electrophoresis on 1% agarose gel (6% formaldehyde). The samples were transferred onto Hybond-N membrane (Amersham Pharmacia Biotech) and probed with a ³²P-labeled ClaI-ClaI fragment (nucleotides 2136-3039 of the chicken LRP cDNA).

Fractionation of Subcellular Membranes and Preparation of Membrane Extract-- Transfected CHO cells were homogenized using a ball bearing homogenizer, and microsomal and plasma membrane fractions were prepared by sequential ultracentrifugation (12). The Triton X-100-soluble membrane extract was prepared either from total cell lysate or from fractionated membranes as described previously (12).

Neuraminidase, Endoglycosidase H, or Endoglycosidase F Digestion of LRP Proteins-- Glycosylation status of LRP was analyzed by sensitivity to Endo H or neuraminidase using fractionated microsomes or plasma membranes or using whole cell lysates as described previously (12). In pulse-chase experiments, confluent cells (35-mm dishes) were labeled with ProMix^TM [³⁵S]methionine and [³⁵S]cysteine (200 µCi/ml), and radiolabeled LRP was immunoprecipitated from whole cell lysates and subjected to Endo H or peptide:N-glycosidase F digestion according to the manufacturer's instructions. The samples were resolved on gradient polyacrylamide gels (3-8%) containing 0.1% SDS (SDS-PAGE), and LRP was visualized by fluorography.

Immunoblot Analysis-- LRP was resolved by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes at 125 V for 6 h under cooling to 10 °C. The LRP -chain and the mutant LRP-RS were detected by immunoblot analysis using an antiserum raised against the C-terminal 17 amino acids of the -chain (designated anti--chain). The LRP -chain was detected using an antiserum against the C-terminal 15 amino acids of the chicken LRP -chain (designated the anti--chain). Immune complexes were visualized by enhanced chemiluminescence.

Endocytosis of ¹²⁵I-Labeled Methylamine-activated ₂M (¹²⁵I-₂M*)-associated LRP-- Confluent cells (35-mm dishes) were incubated with 1 nM ¹²⁵I-₂M* (on ice) for 4 h. After washing with ice-cold phosphate-buffered saline (PBS) containing 10 mg/ml bovine serum albumin, cells were incubated in PBS at 37 °C for 0-10 min. At the indicated times, the dishes were placed on ice, and PBS was removed and replaced with ice-cold F-12 containing pronase (0.3%, w/v). After a 1-h incubation on ice, cells were collected by centrifugation (14,000 × g, 5 min) in a microcentrifuge. Radioactivity associated with the supernatant (pronase-released cell surface ¹²⁵I-₂M*) or cell pellet (internalized ¹²⁵I-₂M*) was quantified. Greater than 90% of the initial radioactivity was recovered during the 10-min experiment.

Internalization and Recycling of Biotinylated LRP-- Internalization studies of biotinylated LRP were performed according to a previously published method (17). Cells (60-mm dishes) were placed on ice and washed twice with ice-cold Krebs-Ringer solution (10 mM sodium phosphate, pH 7.4, 128 mM NaCl, 1.25 mM CaCl₂, 1.25 mM MgSO₄). Cell surface proteins were labeled with 0.5 mg/ml of EZ-Link^TM NHS-SS-Biotin (Pierce) at 0 °C for 1 h. After labeling, cells were incubated with 1 ml of Krebs-Ringer solution (37 °C) for the indicated times. (One dish was kept on ice as the untreated control.) After incubation, cells were treated with or without glutathione solution (15 mg/ml in 1 ml of ice cold Krebs-Ringer solution containing 10% FBS) for 30 min. After the removal of glutathione solution, cells were washed once with ice-cold PBS containing iodoacetamide (5 mg/ml) and twice with PBS alone. Cells were collected into a lysis buffer (Tris-maleate, pH 6.0, Triton X-100 (1.4%, v/v), and phenylmethylsulfonyl fluoride (0.015%, w/v)) in microcentrifuge tubes and mixed for 2 h at 4 °C. The cell lysates were pretreated with protein A-bacterial adsorbent (ICN Canada) prior to immunoprecipitation of LRP with polyclonal antibodies raised against affinity-purified human LRP (see below). The immune complexes were absorbed onto protein A-Sepharose (Amersham Pharmacia Biotech) followed by washing with 1.4% Triton X-100 and dissolved in sample buffer (10 mM Tris-glycine, pH 8.3, 8 M urea, 2% (w/v) SDS, 10% (v/v) glycerol). Bound proteins were resolved by SDS-PAGE (3-8% gels) and transferred to polyvinylidene difluoride membranes. Biotinylated LRP was visualized using horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Preparation of Polyclonal Antibodies against LRP-- Human LRP was affinity-purified from mammary adipose tissue. The tissue was homogenized with extraction buffer (1% octyl--D-glucopyranoside in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 2 mM CaCl₂, 5 mM leupeptin, and 0.1 mg/ml phenylmethylsulfonyl fluoride) (18), and the homogenate supernatant was applied to a glutathione S-transferase/RAP-glutathione Sepharose column (19). The column was washed with extraction buffer, and the bound LRP was eluted using 50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, and 1% octyl--D-glucopyranoside, pH 3.6. The purified LRP was used to immunize New Zealand White rabbits to raise antiserum according to standard procedures (20). The resulting antiserum reacts with both human and chicken LRP.

Other Methods-- The PEA toxicity assay was performed using stably transfected cells as described previously (12). Proteins were determined by the bicinchoninic acid method (Pierce) according to the manufacturer's instructions. Purification, activation, iodination, and uptake/degradation of ₂M* were performed according to procedures reported previously (12).

	RESULTS

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Generation and Expression of Processing Mutant LRP-RS-- The mutant LRP-RS was created by changing the furin cleavage site RNRR³⁹⁴² to RNRS (Fig. 1A), simulating a naturally occurring mutation found in the insulin receptor (10). Nucleotide sequences encoding the mutated furin cleavage site were verified by DNA sequencing (Fig. 1B). Stably transfected cells expressing either LRP-wt or LRP-RS were generated using LRP-null CHO cells (15). Respective stable cell lines were selected that exhibited similar level of LRP expression as judged by immunoblot analysis of total cell lysates (Fig. 1C) and by Northern blotting and metabolic labeling of LRP (data not shown). As expected, expression of LRP-wt led to detection of the processed 515-kDa -chain and 85-kDa -chain that reacted with respective anti-- (Fig. 1C, left) and anti--chain (Fig. 1C, right) antibodies. In contrast, the expression of LRP-RS yielded the unprocessed 600-kDa full-length proreceptor that reacted with both anti-- and anti--chain antibodies (Fig. 1C, right and left). A small amount of unprocessed LRP-wt (600-kDa) that reacted with anti--chain antibody was detectable in LRP-wt transfected cells (Fig. 1C, right). The slight difference in the molecular mass between LRP-RS and the full-length LRP-wt (Fig. 1C, right) was attributable to different glycosylation status between these two proteins (see below). The lack of any secondary cleavage of LRP indicated that no other proteolytic cleavage sites were introduced into LRP by mutagenesis. These results demonstrate that replacing Arg³⁹⁴² with Ser effectively abolishes furin-mediated post-translational endoproteolysis of LRP.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Mutagenesis and expression of recombinant LRP variants in transfected LRP-null cells. A, schematic diagram of site-specific mutagenesis using parental expression plasmid pcLRP-100 (12). Positions of the translation start (ATG) and stop (TAG) codons are indicated by closed triangles, and the processing site is represented by an upward arrow. The sequence of RNRR in the wild-type LRP (pcLRP-wt) was changed into RNRS in the processing mutant LRP (pcLRP-RS). A PvuI restriction site (underlined) was introduced in the mutant construct. B, sequencing gel of the resulting expression plasmid DNA. C, immunoblot analysis of LRP variants stably expressed in LRP-null cells using anti-- or anti--chain antibody. cLM, chicken liver membrane extracts; mock, cells transfected with pSV2neo.

Cell Surface Presentation of LRP and Restored PEA Sensitivity-- Plasma membrane was isolated from transfected cells and treated with neuraminidase. Like - and -chains of LRP-wt, the unprocessed LRP-RS proreceptor was sensitive to neuraminidase treatment (Fig. 2A). Thus, abolishing post-translational processing does not alter trafficking of LRP from the ER, through the trans-Golgi compartment, to the plasma membrane.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Functional expression of LRP-wt and mutant LRP-RS on cell surface. A, neuraminidase digestion of plasma membrane LRP. Plasma membranes were isolated from cells expressing wild-type (LRP-wt) or the processing mutant LRP (LRP-RS) and treated with (+) or without () neuraminidase as described under "Experimental Procedures." Samples were resolved by SDS-PAGE, and LRP proteins were detected by immunoblotting as in Fig. 1C. B, susceptibility to Pseudomonas exotoxin A of LRP-transfected cells. Parental cells (LRP-null) and cells expressing wild-type (LRP-wt) or processing mutant LRP (LRP-RS) were plated, treated with PEA, and assayed for [35S]methionine/cysteine incorporation as described previously (12). Normal CHO-K1 cells expressing endogenous LRP were used as a control.

Endocytosis of ₂M in LRP-transfected Cells-- Expression of either LRP-wt or LRP-RS in LRP-null cells also restored the ability of the cells to bind and degrade ₂M*. The RAP-inhibitable binding of ¹²⁵I-₂M* at 4 °C was similar between LRP-wt- and LRP-RS-transfected cell lines at three concentrations (i.e. 0.5, 1.0, and 2.5 nM) of the ligand (Fig. 3A). Binding of ¹²⁵I-₂M* to the transfected cells was about 2.5-fold greater than that to normal CHO-K1 cells (Fig. 3A). Degradation of ¹²⁵I-₂M* in cells expressing the unprocessed LRP-RS proreceptor was as efficient as that in LRP-wt-expressing cells (Fig. 3B). These results suggest that the LRP-mediated endocytosis and lysosomal degradation of ₂M* are independent of LRP processing.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Surface binding and degradation of 125I-2M* in LRP-transfected cells. A, RAP-inhibitable binding of 125I-2M* to parent cell (Null), normal CHO-K1 (K1), cells expressing wild-type LRP (wt), or cells expressing processing mutant LRP (RS). Total binding was determined using confluent monolayers (six-well plates) incubated on ice for 4 h with the indicated concentration of 125I-2M*. Nonspecific binding was determined in incubations performed in the presence of 100 nM RAP. Data points are the RAP-inhibitable binding, calculated as the difference between total and nonspecific binding (average of two independent experiments). B, degradation of 125I-2M*. Confluent monolayers (six-well plates) were incubated with 1 nM 125I-2M* at 37 °C. At the indicated times, the medium was collected and proteins were precipitated with 15% trichloroacetic acid. Trichloroacetic acid-soluble, noniodide radioactivity was determined in the aqueous phase following treatment of the 15% trichloroacetic acid supernatant with 40% KI and H2O2 and extraction of liberated iodine with chloroform. Data points are RAP-inhibitable degradation, determined by subtracting total degradation counts from those determined in the presence of 100 nM RAP.

The Endocytic Rate of LRP-- The endocytic rate of surface-bound ₂M* was determined using two approaches. In the first approach, cells were incubated with ¹²⁵I-₂M* at 0 °C and then switched to 37 °C. Internalization of surface-bound ¹²⁵I-₂M* was monitored by measuring the acquisition of resistance of ¹²⁵I-₂M* to exogenous pronase (Fig. 4A). At the beginning of temperature switch (time zero), ~70% of the total ¹²⁵I-₂M* was surface-bound (i.e. 30% pronase-resistant). After incubation at 37 °C for 5 min, 80% of total ¹²⁵I-₂M* became pronase-resistant (Fig. 4A). There was no difference between LRP-wt and LRP-RS in gaining pronase resistance, suggesting that efficient internalization of ¹²⁵I-₂M* can be achieved through both receptors.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Endocytosis of 125I-2M* in LRP-transfected cells. A, 125I-2M* (1 nM) was bound to nearly confluent monolayers (35-mm dishes) for 4 h on ice and then incubated with warm (37 °C) PBS for the indicated time. The dishes were placed on ice and treated with pronase as described under "Experimental Procedures." The pronase-resistant and -sensitive 125I-2M* was quantified and expressed as a percentage of the initial 125I-2M* bound (wild type, 2.3 × 105 cpm/mg of cell protein; RS mutant, 1.8 × 105 cpm/mg of cell protein). Total radioactivity (cells plus supernatant) was constant throughout the experiment. B, In/Sur analysis. Cells were labeled with 2 nM 125I-2M* (37 °C) and placed on ice at the indicated times. The cell surface (Sur) and internalized (In) 125I-2M* were quantified following pronase digestion for the calculation of Ke. Similar results were obtained when the experiment was repeated using 1 nM 125I-2M*.

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Internalization and Recycling of LRP-- Constitutive cycling of biotinylated LRP between the cell surface and the endosomal compartment was analyzed using two protocols. First, cell surface LRP was labeled with a thiol-cleavable biotin linker (sensitive to glutathione) at 0 °C, and turnover of the surface LRP was followed at 37 °C (Fig. 5A). Initially, nearly all of the biotinylated LRP proteins were on the cell surface, as shown by their complete sensitivity to glutathione (time 0). After incubation at 37 °C for 5 min, approximately 60% of the surface LRP was internalized, therefore gaining glutathione resistance. No significant difference in the constitutive LRP internalization was observed between LRP-wt and LRP-RS. The internalized LRP remained glutathione-resistant for 30 min, suggesting that at least 30 min is required for recycling of LRP back to the plasma membrane. There was no change in the level of total cellular LRP-wt or LRP-RS during the entire course of the experiments, as demonstrated by immunoblotting using anti--chain or anti--chain antibodies (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Recycling of LRP-wt and mutant LRP-RS in transfected cells. A, cells (60-mm dishes) were labeled with NHS-SS-Biotin on ice and then incubated at 37 °C for the indicated times. After treatment with glutathione to cleave the biotin moiety of cell surface LRP, cells were lysed, and LRP was recovered by immunoprecipitation. Samples were resolved by SDS-PAGE (on a 3-8% gradient gel), and biotinylated LRP was visualized using horseradish peroxidase-conjugated streptavidin. Biotinylated LRP was quantified by scanning densitometry. The glutathione-resistant signal at each time point is expressed as a percentage of the untreated control (NT). B, cells were labeled with NHS-SS-biotin at 37 °C for 2 h and treated with glutathione (0 °C) to remove the biotin moiety from cell surface LRP. Monolayers were then incubated at 37 °C for the indicated time, and the sensitivity of biotinylated LRP to glutathione was analyzed at each point as described above. Repetition of the experiments yielded essentially the same results.

Post-translational Stability and Intracellular Trafficking of LRP-- The effect of abolishing processing on post-translational stability of LRP was determined by pulse-chase analysis. At the end of 1 h of pulse, only the proreceptor forms of LRP-wt and LRP-RS were detected (Fig. 6A, band a). Both of the proreceptors were located predominately within the ER, since the carbohydrate moieties of the proreceptors were sensitive to digestion with Endo H (Fig. 6B, bands a and a'). Conversion of the Endo H-sensitive proreceptors into Endo H-resistant species (from band a to band b) occurred between 1 and 2 h of chase. There was no difference between LRP-wt and LRP-RS in the time required to attain Endo H resistance, suggesting that the rate of transit between the ER and Golgi apparatus was independent of LRP processing. The half-life of the post-ER form of mutant LRP-RS (i.e. band b) as estimated between 4 h (at the peak) and 15 h of chase, was approximately 10 h, similar to that of LRP-wt (i.e. band c in Fig. 6A, Endo H-resistant in Fig. 6B).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Post-translational stability and intracellular trafficking of LRP-wt and mutant LRP-RS. A, cells were pulse-labeled with [35S]methionine and [35S]cysteine for 1 h and incubated in chase medium for up to 15 h. At the indicated times, LRP was immunoprecipitated from cell lysates, and aliquots of sample were resolved by SDS-PAGE. The position labeled a represents proreceptor (i.e. full-length LRP-wt or LRP-RS) with simple carbohydrate modification, whereas that labeled b represents proreceptor containing complex carbohydrate modification. The position labeled c indicates the processed LRP-wt -chain. B, immunoprecipitated LRP samples were digested with Endo H or peptide:N-glycosidase F. Simple glycosylated proreceptors (a in lanes C (control)) are Endo H-sensitive (a' in lanes H (Endo H)), whereas complex glycosylated proreceptors (b in lanes C and H) and the processed LRP-wt -chain (c) are Endo H-resistant. Note the persistence of the Endo H-sensitive proreceptor (a, a') of mutant LRP-RS. Lane F, peptide:N-glycosidase F treatment.

	DISCUSSION

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Since the discovery of proteolytic cleavage of LRP in 1990 by Herz et al. (7), little has been learned about the biological function of this post-translational processing event. Here, using an expression system, we examine the effect of a single amino acid substitution at the processing site of chicken LRP on its endocytic functions and intracellular trafficking. We have shown that the proteolytic processing can be completely blocked by replacing Arg³⁹⁴² with Ser at the furin cleavage site (RHRR³⁹⁴²), clearly indicating that LRP processing indeed occurs at the consensus furin cleavage site during its maturation and transport to the cell surface. Data obtained from the present studies have shown that the processing mutant LRP-RS is transported onto the plasma membrane (Fig. 2A), presumably via the classical ER-to-Golgi trafficking. In addition, the mutant LRP-RS appears to be fully functional, as demonstrated by its ability to bind the three ligands examined (i.e. ₂M*, RAP, and PEA). Expression of LRP-RS can, as effectively as LRP-wt, restore the sensitivity of LRP-deficient cells to PEA (Fig. 2B) as well as reconstitute their ability to bind, internalize, and degrade ₂M* in a RAP-inhibitable fashion (Fig. 3). Furthermore, the rates of endocytosis and recycling of LRP-RS, either in ₂M*-bound form (Fig. 4) or else as an unoccupied receptor (Fig. 5), are nearly identical to those of LRP-wt. Therefore, the present results, together with previous observations of the truncated minireceptors (11, 12), all indicate that the unprocessed receptors exhibit endocytic functions indistinguishable from LRP-wt, providing strong evidence that post-translational endoproteolysis is not essential for endocytosis.

While mutational analysis detects no involvement of LRP processing in endocytosis, the possibility that processing is involved in other cellular functions remains to be tested. Recently, Bu et al. (22) have reported that the cytosolic tail of LRP is phosphorylated in a neuronal cell line (GT1-1) transfected with the receptor for nerve growth factor and that the phosphorylation increases upon the growth factor treatment. The functional role of phosphorylation of the cytosolic tail of LRP is yet unclear. Early studies with a processing mutant form of insulin receptor showed that the unprocessed receptor expressed in transfected CHO cells bound insulin normally but was inefficiently phosphorylated (23, 24). Therefore, it will be of interest to determine in future studies the effect of abolishing processing on phosphorylation of LRP. Since the cytoplasmic domain of integral membrane proteins plays a major role in sorting and targeting, phosphorylation may be part of signaling pathways responsible for acute regulation and distribution of LRP between different intracellular pools (22). The existence of an endosomal "storage" pool of LRP has been suggested in studies with rat adipocytes, in which a rapid and transient mobilization of LRP from the intracellular pool to cell surface was observed when the cells were treated with insulin (25). Recently, we have obtained preliminary experimental evidence showing that the insulin-induced cell surface presentation of LRP is sensitive to inhibitors of phosphatidylinositide 3-kinase in 3T3-L1 adipocytes.² Thus, it will also be of interest in future studies to determine if LRP processing plays a role in the acute redistribution of LRP between endosomes and plasma membranes.

An important observation made in this study is the delayed LRP transport out of ER caused by the single amino acid substitution at the furin cleavage site. The prolonged ER retention displayed by the mutant LRP-RS is not likely attributable to an artifact of overexpression of the mutant receptor, since the level of protein synthesis between LRP-RS and LRP-wt was similar (Fig. 6). The mechanism responsible for the delayed ER exit of LRP-RS is unclear, but some possibilities may be considered. It has been shown in human glioblastoma U87 cells (27, 28) and in transgenic mice (29) that the molecular chaperone RAP confers proper folding of LRP and prevents ligand-induced aggregation of the receptor. Five RAP-binding sites have been identified within the class A repeats of human LRP (30), but it is not known if there are additional RAP-binding sites within sequences other than the class A repeats. It is possible that introducing the Arg³⁹⁴²-to-Ser substitution may have impaired the affinity of nascent LRP for RAP. Interaction of LRP with RAP within the secretory pathway was not determined in this study. However, we did not find the retained mutant LRP-RS to be aggregated or rapidly degraded within the ER. The half-life of mutant LRP-RS that was retained in the ER (i.e. remained sensitive to Endo H) was 12-15 h (Fig. 6). Therefore, it is unlikely that the retarded ER exit of mutant LRP-RS was a consequence of impaired association with RAP. Another possibility for the delayed mutant LRP transport out of the ER is that the sequence RXRR acts as a recognition site for the ER quality control system. Studies with renin, a secretory protein derived from proteolytic cleavage of the precursor prorenin, have shown that the presence of a protease processing site per se within prorenin is essential for its sorting and secretion (31). Exposure of the potential proteolytic cleavage site (RXRR) within LRP may be essential for efficient ER exit in addition to furin cleavage. Conclusive evidence of this functionality of the LRP processing site will require further definitive studies.

Recently, it was reported that the 515-kDa -chain of human LRP could occur as a soluble form in the plasma (32), raising the possibility that processing may play a role in shedding. In CHO cells transfected with either wild type or the processing mutant LRP, we could not detect any -chain or LRP-derived fragments in the media (data not shown). These results suggest that processing per se is not sufficient for the release of -chain from cells in culture. However, when a cDNA construct containing an engineered premature stop codon at the carboxyl terminus of the RNRR sequence was expressed in CHO cells, we found that the encoded LRP -chain accumulated in the culture medium.³ Thus, soluble LRP fragments found in the plasma may arise from proteolysis at the cell surface (independent of furin cleavage) and subsequent dissociation of the -chain from the transmembrane fragment.

In summary, post-translational processing of the chicken LRP does not grossly affect its endocytic functions in transfected CHO cells. However, transport of LRP out of the ER appears to be extremely sensitive to a single amino acid substitution at the endoproteolytic cleavage site. Mechanisms that are responsible for the retarded ER exit of the processing mutant form of LRP are currently under investigation.