The YXXL Motif, but Not the Two NPXY Motifs, Serves as the Dominant Endocytosis Signal for Low Density Lipoprotein Receptor-related Protein

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 275, No. 22, Issue of June 2, pp. 17187–17194, 2000 Printed in U.S.A. The YXXL Motif, but Not the Two NPXY Motifs, Serves as the Dominant Endocytosis Signal for Low Density Lipoprotein Receptor-related Protein* Received for publication, January 19, 2000, and in revised form, March 21, 2000 Published, JBC Papers in Press, March 28, 2000, DOI 10.1074/jbc.M000490200 Yonghe Li‡, Maria Paz Marzolo§, Peter van Kerkhof¶, Ger J. Strous¶, and Guojun Bu‡i From the ‡Departments of Pediatrics, and Cell Biology and Physiology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri 63110, the §Department of Biology, University of Chile, Santiago, Chile, and the ¶Department of Cell Biology, Utrecht University, Utrecht, The Netherlands All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation. The low density lipoprotein (LDL)1 receptor-related protein (LRP) is a member of the LDL receptor (LDLR) gene family, which consists of at least six known cell surface receptors: LDLR itself, LRP, the very low density lipoprotein receptor (VLDLR), megalin/LRP-2, apolipoprotein E receptor-2 (apoER2)/LR8B, and LR11 (1– 4). LRP is synthesized as a 600kDa single-chain precursor, which undergoes post-translational proteolytic processing within the trans-Golgi compart- * This work was supported by National Institutes of Health Grants NS37525 and HL59150 (to G. B.) and Fondo Nacional de Ciencia y Technologia Grant 1990600 and Departmento de Investigacion y Desarrollo Grant I007-98/2 (to M. P. M.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i To whom correspondence should be addressed: Dept. of Pediatrics, Washington University School of Medicine, Box 8116, One Children’s Pl., St. Louis, MO 63110. Tel.: 314-454-2726; Fax: 314-454-2685; Email: bu@kids.wustl.edu. 1 The abbreviations used are: LDL, low density lipoprotein; CHO, Chinese hamster ovary; LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; apoE, apolipoprotein E; apoER2, apolipoprotein E receptor-2; VLDLR, very low density lipoprotein receptor; scuPA, single chain urokinase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; BSA, bovine serum albumin; ER, endoplasmic reticulum. This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ment by the endopeptidase furin (5, 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 (5). The 515-kDa subunit contains all of the putative ligand-binding domains consisting of 31 copies of the complement-type ligandbinding repeats arranged in four clusters of 2, 8, 10, and 11 (1, 2). The 85-kDa subunit contains a single transmembrane domain and the cytoplasmic tail. LRP is widely expressed in adult tissues, most abundantly in the liver and the brain (1, 7, 8). To date, more than 10 structurally and functionally distinct ligands have been identified, including a2-macroglobulin, apoE/ lipoproteins, lipoprotein lipase, tissue-type plasminogen activator, and urokinase (1, 2, 9). Ligand interactions with LRP can be antagonized by a 39-kDa receptor-associated protein (RAP). The recombinant form of RAP has been used extensively in the study of ligand-receptor interactions. Under physiological conditions, however, RAP functions intracellularly as a molecular chaperone for LRP and facilitates LRP folding and trafficking within the secretory pathway (9). Increasing evidence has shown that LRP plays important roles in lipoprotein remnant catabolism (10), protease regulation (11), cell migration (12, 13), neuronal process outgrowth (14), and the pathogenesis of Alzheimer’s disease (15, 16). Cell surface receptors that traffic between the plasma membrane and endocytic compartments contain signals within their cytoplasmic tails that allow their efficient recruitment into endocytic vesicles. To date, four classes of endocytosis signals have been identified that target surface proteins to clathrincoated vesicles. Tyrosine-based signals within NPXY or YXXØ sequence (where X can be any amino acid and Ø is an amino acid with a bulky hydrophobic group) were initially identified in LDLR and transferrin receptor, respectively (17). In many cases these signals are constitutively active, i.e. receptors undergo continuous rounds of endocytosis and recycling independent of ligand binding. The di-leucine motif is another well known endocytosis motif that is present within many transmembrane cell surface proteins (18). The third type of endocytosis signal is serine phosphorylation. For several members of the family of G protein-coupled receptors, ligand-induced phosphorylation of serine residues serves as a signal for receptor endocytosis (19). In CD4 and CD3g, the di-leucine motif acts in cooperation with a neighboring phosphorylated serine residue to mediate endocytosis (20, 21). Finally, the attachment of ubiquitin moieties has been recently identified as another regulator of the endocytosis of several membrane receptors (22, 23). A common characteristic of the LDLR family members is that they all contain at least one copy of the NPXY sequence 17187 17188 LRP Endocytosis Signals within their cytoplasmic tails. For LDLR, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits (24). Therefore, it is generally believed that the NPXY sequences in these receptors categorically serve as endocytosis signals. More recently, Herz and colleagues (25–27) have demonstrated that two cytoplasmic adaptor proteins, mammalian Disabled-1 and FE65, interact with NPXY motifs in the cytoplasmic tails of LRP, LDLR, VLDLR, and ApoER2, and that VLDLR and ApoER2 function as obligate components in the Reelin/Disabled-mediated neuronal migration pathway. These data suggest that the NPXY motifs within the tails of the LDLR family members may function not only as endocytosis signals but also as binding motifs for cellular components, which are involved in signal transduction. The tail of LRP consists of 100 amino acid residues, and contains multiple potential endocytosis motifs including two NPXY motifs, one YXXØ motif, and two di-leucine motifs. In this study, the five potential endocytosis signals are termed, in their relationships to the transmembrane domain, as proximal NPXY, proximal di-leucine, distal NPXY, YXXL, and distal di-leucine (see Fig. 1). The primary aim of this study is to define endocytosis signal(s) within the LRP tail. Here, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also show that the distal di-leucine motif contributes to LRP endocytosis, and its function is independent of the YXXL motif. These data suggest that each member of the LDLR family may utilize different potential signal(s) within their cytoplasmic tails for receptor-mediated endocytosis. EXPERIMENTAL PROCEDURES Materials—Human recombinant RAP was expressed in a glutathione S-transferase expression vector and isolated as described previously (28). scuPA was kindly provided by G. F. Vovis of Collaborative Research (29). All tissue culture media, serum, and plasticware were from Life Technologies, Inc. Non-enzymatic cell dissociation solution was from Sigma. Monoclonal anti-HA antibody has been described before (30). Goat anti-mouse IgFITC was from Becton Dickinson. Cy3-Goat anti-mouse IgG was from Sigma. Quantum Simply Cellular microbead standard was from Flow Cytometry Standards Corp., San Juan, Puerto Rico. Peroxidase-labeled anti-mouse antibody and ECL system were from Amersham Pharmacia Biotech. Immobilon-P transfer membrane was from Millipore. Rainbow molecular weight markers were from Bio-Rad. Carrier-free Na125I was purchased from NEN Life Science Products. Cell Culture and Transfection—The LRP-null Chinese hamster ovary (CHO) cell line (kindly provided by David FitzGerald, National Institutes of Health; see Ref. 31) was cultured in Ham’s F-12 medium as described (31). Stable transfection into LRP-null CHO cells was achieved by transfection of 30 mg of plasmid DNA in 10-cm dishes by using a calcium phosphate precipitation method (32). Stable transfectants were selected using 700 mg/ml G418 and maintained with 350 mg/ml G418. Construction of LRP Minireceptor and Site-directed Mutagenesis— The construction of the membrane-containing minireceptor of LRP (see Fig. 1, A and C) via polymerase chain reaction was performed essentially as described previously (30, 33). Site-directed mutagenesis was carried out using the QuickChange mutagenesis kit (Stratagene) according to the manufacturer’s instructions. All oligonucleotides were synthesized at the Washington University School of Medicine Protein Chemistry Laboratory. Western Blotting of LRP Minireceptors—Stably transfected CHO cells were lysed with 0.5 ml of lysis buffer (phosphate-buffered saline containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Equal quantities of protein were subjected to SDSPAGE (6%) under reducing conditions. Following transfer to polyvinylidene difluoride membrane, successive incubations with anti-HA antibody and horseradish peroxidase-conjugated goat anti-mouse IgG were carried out for 60 min at room temperature. The immunoreactive proteins were then detected using the ECL system. Films showing immunoreactive bands were scanned using a Kodak Digital Science DC120 Zoom digital camera and analyzed with Kodak Digital Science1D image analysis software. Flow Cytometric Analysis of Cell Surface LRP Minireceptors—For cell surface LRP minireceptor analysis, living cells were used (34). Briefly, CHO cells were detached by incubation with non-enzymatic cell dissociation solution. Successive incubations with affinity-purified anti-HA IgG (25 mg/ml) and goat anti-mouse IgFITC were carried out at 4 °C for 45 min. Background fluorescence intensity was assessed in the absence of primary monoclonal antibody. The antibody binding capacities were evaluated from the standardized Quantum Simply Cellular bead calibration plot (35). The bead standards consist of four populations of microbeads coated with goat anti-mouse antibody, which bind different numbers of mouse IgG monoclonal antibody molecules (5686, 18,329, 50,908, and 150,477 molecule binding capacities) in addition to a blank population. The beads were stained in the same way as the CHO cells. Protein Iodination—RAP and scuPA (50 mg) were iodinated by using the IODO-GEN method as described previously (36). Kinetic Analysis of Endocytosis—Kinetic analysis of endocytosis was performed according to previously published methods (37, 38). Stably transfected CHO cells were plated in 12-well plates at a density of 2 3 105 cells/well and used after overnight culture. Cells were rinsed twice in ice-cold ligand binding buffer (minimal Eagle’s medium containing 0.6% BSA), and 125I-RAP was added at 5 nM final concentration in cold ligand binding buffer (0.5 ml/well). The binding of 125I-RAP was carried out at 4 °C for 30 min with gentle rocking. Binding of 125I-RAP was specific, i.e. the addition of 100-fold excess unlabeled RAP inhibited binding by 90 –95%. Unbound ligand was removed by washing cell monolayers three times with cold binding buffer. Ice-cold stop/strip solution (0.2 M acetic acid, pH 2.6, 0.1 M NaCl) was added to one set of plates without warming up and kept on ice. The remaining plates were then placed in a 37 °C water bath, and 0.5 ml of ligand binding buffer prewarmed to 37 °C was quickly added to the well monolayers to initiate internalization. After each time point, the plates were quickly placed on ice and the ligand binding buffer was replaced with cold stop/strip solution. Ligand that remained on the cell surface was stripped by incubation of cell monolayers with cold stop/strip solution for a total of 20 min (0.75 ml for 10 min, twice) and counted. Cell monolayers were then solubilized with low SDS lysis buffer (62.5 mM Tris-HCl, pH 6.8, 0.2% SDS, 10% v/v glycerol) and counted. The sum of ligand that was internalized, plus that which remained on the cell surface after each assay was used as the maximum potential internalization. The fraction of internalized ligand after each time point was calculated and plotted. Analyses of LRP Ligand Binding Activity and Ligand Degradation Efficiency—Cells (2 3 105) were seeded into 12-well dishes 1 day prior to assays. Assay buffer (minimal Eagle’s medium containing 0.6% BSA with 5 nM radioligand, 0.6 ml/well) was added to cell monolayers, in the absence or the presence of unlabeled 500 nM RAP, followed with incubation for 1 h at 4 °C. Thereafter, overlying buffer containing unbound ligand was removed, and cell monolayers were washed and lysed in low SDS lysis buffer and counted. Ligand degradation efficiency was measured using the methods as described (24, 39). Briefly, 2 3 105 cells were seeded into 12-well dishes 1 day prior to assays. Pre-warmed assay buffer was added to cell monolayers, in the absence or the presence of unlabeled 500 nM RAP, followed with incubation for 4 h at 37 °C. Thereafter, the medium overlying the cell monolayers was removed and proteins were precipitated by addition of BSA to 10 mg/ml and trichloroacetic acid to 20%. Degradation of radioligand was defined as the appearance of radioactive fragments in the overlying medium that were soluble in 20% trichloroacetic acid. The protein concentrations of each cell lysates were measured in parallel dishes that did not contain LRP ligands. The ligand degradation efficiency is the value of degraded ligand (cpm/mg cell protein) divided by the number of cell surface LRP minireceptors (as determined by flow cytometry, and calculated relative to wild type mLRP4T100). Immunofluorescence Microscopy—Stably transfected CHO cells expressing various proteins were grown on glass coverslips, fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The cells were then incubated for 30 min with anti-HA antibody followed by a 30-min labeling with Cy3 goat anti-mouse IgG. Confocal microscopy was performed on a Bio-Rad MRC 1024 model using a Zeiss 633 (nominal aperture, 1.4) oil immersion lens. RESULTS Endocytosis Rate of LRP Minireceptors with Truncated Tails—The very large size of LRP (;600 kDa) limits molecular LRP Endocytosis Signals 17189 FIG. 1. Endocytosis rate of LRP minireceptors with truncated cytoplasmic tails. A, schematic representation of mLRP4T100. mLRP4T100 is depicted in comparison to the full-length LRP molecule. The four putative ligand-binding domains are labeled with Roman numerals I, II, III, and IV. B, the sequence of LRP cytoplasmic tail. The first amino acid following the transmembrane domain is numbered 1. C, schematic representation of potential endocytosis signals within the tails of mLRP4T100, and its deletion variants. D, effects of tail truncation on LRP minireceptor-mediated endocytosis. LRP-null CHO cells stably transfected with mLRP4T100, and its deletion variants were incubated with 5 nM 125I-RAP at 4 °C for 30 min, and then shifted to 37 °C for the indicated times. The amounts of ligand internalized as the fraction of the total cell-associated ligand (the sum of the internalized ligand plus the ligand remaining on the cell surface at the end of the assay; see “ Experimental Procedures” for further explanation) are plotted against time. Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of three such experiments performed with similar data. manipulations at the cDNA level and the expression of this protein via transfection. Thus, we generated an LRP minireceptor that mimics the function and trafficking of LRP (Fig. 1A). This LRP minireceptor encodes residues 2462– 4525 of the full-length LRP (7), which includes the fourth cluster of ligandbinding repeats through the carboxyl terminus of the receptor (designated mLRP4T100, with “m” representing membranecontaining, “4” representing the fourth cluster of ligand-binding repeats, “T” representing cytoplasmic tail, and “100” representing the 100 amino acid residues within the LRP tail). To facilitate immunoprecipitation and Western blot analysis, an HA epitope was included near the amino terminus of mLRP4T100. Our studies have demonstrated that this LRP minireceptor mimics the function and trafficking of full-length endogenous LRP (30). Fig. 1B shows the sequence of the cytoplasmic tail of LRP with the first amino acid following the transmembrane domain numbered as 1. Using LRP-null CHO cells (31), we generated stably transfected cell lines expressing LRP minireceptors with different potential endocytosis signals after truncation (Fig. 1C), and measured the endocytosis rate of these truncated LRP minireceptors. Our previous studies have shown that the fourth ligand-binding domain of LRP binds RAP with high affinity (30, 32, 33). Fig. 1D shows the effects of tail truncation on LRP minireceptor-mediated endocytosis. As shown in the figure, the stable cell line bearing mLRP4T100 rapidly internalized 125I-RAP, with a half-time of less than 30 s. In contrast, 125 I-RAP was poorly internalized by cells expressing either mLRP4Ttailess or mLRP4T25, while the endocytosis rates of mLRP4T59 and mLRP4T72 were variably greater than those of mLRP4Ttailess and mLRP4T25. These results suggest each of the potential endocytosis motifs (NPXY, YXXØ, and di-leucine) within the LRP tail may contribute to LRP endocytosis. The YXXL Motif, but Not the Two NPXY Motifs, Serves as the Dominant Endocytosis Signal for LRP—Based on the above results, we generated eight LRP minireceptors with specific mutations in the cytoplasmic tail based on mLRP4T100 (Fig. 2A). These mutations were targeted to each of the putative endocytosis signals, which allowed us to evaluate the contribution of these signals in LRP endocytosis. Fig. 2B shows the endocytosis rates of wild type mLRP4T100 and its proximal NPXY motif mutants. Alteration of the asparagine or the tyrosine to alanine resulted in only a slight decrease in RAP internalization, with the initial endocytosis rates being indistinguishable from that of mLRP4T100. These results indicate that the proximal NPXY motif is not important for LRP endocytosis. Fig. 2C shows the endocytosis rates of wild type mLRP4T100 and its distal NPXY motif mutants. As the phenylalanine is the important residue within the FXNPXY motif for LDLR endocytosis (24), we also mutated this residue to alanine. Replacement of the phenylalanine or the asparagine by alanine resulted in only a slight decrease in RAP internalization, with the initial endocytosis rates being indistinguishable from that of mLRP4T100. In contrast, alteration of the tyrosine to alanine resulted in a dramatic decrease in RAP internalization. At 15, 30, and 60 s, this mutant internalized 3%, 5%, and 12%, respectively, of the total cell-associated 125I-RAP, corresponding to a 80 –90% impairment in endocytosis relative to wild type mLRP4T100. These results indicate that the tyrosine residue at position 63 is critical for LRP endocytosis. Because the tyrosine within the distal NPXY motif is also within the YXXL motif, the decreased endocytosis rate of mLRP4T100(Y63A) may have resulted from the mutation of the YXXL motif. Fig. 2D shows the endocytosis rates of wild type mLRP4T100 and its YXXL motif mutants. As shown in the figure, the endocytosis rate of mLRP4T100(L66A) was significantly decreased, and was indistinguishable from that of mLRP4T100(Y63A). Taken together, these results clearly demonstrate that the YXXL motif, but not the distal NPXY motif, serves as the major endocytosis signal for LRP. 17190 LRP Endocytosis Signals FIG. 2. Endocytosis rate of LRP minireceptors with site-mutated cytoplasmic tails. A, schematic representation of potential endocytosis signals within the tails of LRP4T100, and its mutants. The introduced amino acid substitutions are shown in bold. B–E, effect of mutations in LRP tail on LRP minireceptor-mediated endocytosis. 125I-RAP internalization in LRP-null CHO cells stably transfected with mLRP4T100, and its mutants was carried out for indicated time points as described in the Fig. 1D legend. Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of three such experiments performed with similar data. B, endocytosis of 125I-RAP by LRP minireceptors with mutations in the proximal NPXY motif. C, endocytosis of 125I-RAP by LRP minireceptors with mutations in the distal NPXY motif. D, endocytosis of 125I-RAP by LRP minireceptors with mutations in the YXXL motif. E, endocytosis of 125IRAP by LRP minireceptors with mutations of the two di-leucine motifs. Distal Di-leucine Motif, but Not the Proximal Di-leucine Motif, Contributes to LRP Endocytosis—The di-leucine motif is another well characterized endocytosis signal that is present in many transmembrane cell surface proteins. Thus, we next compared the endocytosis rate of mLRP4T100 with that of its di-leucine motif mutants (Fig. 2E). Mutation of both leucines to alanines in the proximal di-leucine motif resulted in little or no change in RAP internalization. However, the mLRP4T100(L86A,L87A) mutant clearly demonstrates a defect in internalization, although not as substantial as mLRP4T100(Y63A) or mLRP4T100(L66A) (Fig. 2, D and E). At 15, 30, and 60 s, mLRP4T100(L86A,L87A) internalized 18%, 30%, and 40%, respectively, of the total cell-associated 125IRAP, corresponding to a 30–40% impairment in endocytosis relative to wild type mLRP4T100. These results clearly demonstrate that the distal di-leucine motif, but not the proximal di-leucine motif, contributes to LRP endocytosis. The YXXL Motif and the Distal Di-leucine Motif Function Independently in LRP Endocytosis—To address whether the YXXL motif and the distal di-leucine motif function independently in LRP endocytosis, we generated a double mutant, mLRP4T100(Y63A,L86A,L87A). The endocytosis rate of this mutant receptor was compared with that for wild type mLRP4T100 as well as mLRP4T100(Y63A), mLRP4T100(L86A,L87A), and mLRP4Ttailess, in stably transfected LRP-null CHO cells. As shown in Fig. 3, the endocytosis rate of mLRP4T100(Y63A,L86A,L87A) was significantly lower than that of mLRP4T100(Y63A). These results thus clearly demonstrate that the YXXL motif and the distal di-leucine motif function independently in LRP endocytosis. FIG. 3. The YXXL motif and the distal di-leucine motif function independently for LRP endocytosis. 125I-RAP internalization in LRP-null CHO cells stably transfected with mLRP4T100, mLRP4T100(L86A,L87A), mLRP4T100(Y63A), mLRP4T100(Y63A,L86A,L87A), and mLRP4Ttailess was carried out for indicated time points as described in the Fig. 1D legend. Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of three such experiments performed with similar data. The Proximal NPXY Motif and the Proximal Di-leucine Motif Function in a Truncated Receptor Tail—In Fig. 2 (B and E), we demonstrated that the proximal NPXY motif and the proximal di-leucine motif exhibit little or no role in LRP endocytosis within the full-length LRP tail. However, comparison of the LRP Endocytosis Signals 17191 FIG. 4. The proximal NPXY motif and the proximal di-leucine motif function in truncated receptor tail. 125I-RAP internalization in LRP-null CHO cells stably transfected with mLRP4T100, mLRP4T59, mLRP4T59(Y29A), mLRP4T59(L43A,L44A), and mLRP4Ttailess was carried out for indicated time points as described in the Fig. 1D legend. Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of three such experiments performed with similar data. endocytosis rate of mLRP4T100 with that of mLRP4T59 indicated that mLRP4T59 is still capable of internalizing RAP (Fig. 1D). To address this question, we generated two mLRP4T100 mutants mLRP4T59(Y29A) and mLRP4T59(L43A,L44A) using mLRP4T59 as the template. Fig. 4 shows the endocytosis rates of the stably transfected CHO cells expressing mLRP4T100, mLRP4T59, mLRP4T59(Y29A), mLRP4T59(L43A,L44A), and mLRP4Ttailess. As shown in the figure, the endocytosis rates of mLRP4T59(Y29A) and mLRP4T59(L43A,L44A) were significantly lower than that of mLRP4T59, indicating that the proximal NPXY motif and the proximal di-leucine motif are functional within the truncated receptor tail. mLRP4T100 Mutants Defective in Endocytosis Accumulate at the Cell Surface—We hypothesized that mLRP4T100 mutants that are defective in endocytosis signals would accumulate at the cell surface. We initially compared the ligand binding activity of wild type mLRP4T100 with those of endocytosis mutants expressed in stably transfected CHO cells. As shown in Fig. 5A, CHO cells expressing wild type mLRP4T100 exhibited a moderate level of cell surface RAP binding, while CHO cells transfected with the pcDNA3 vector exhibited only ;10% of RAP binding compared with those transfected with mLRP4T100. The residual RAP binding to pcDNA3-transfected cells is likely mediated by cell surface heparan sulfate proteoglycan (40). Interestingly, CHO cells expressing mLRP4T100 mutants defective in endocytosis signals exhibited significant increase in cell surface RAP binding activity. To analyze whether the increase of RAP binding to CHO cells expressing endocytosis mutants is due to an accumulation of mature minireceptor on the cell surface, we examined the steady-state distribution of various forms of minireceptors via Western blotting analysis using anti-HA antibody (Fig. 5B). For mLRP4T100 and its mutants, two distinct bands are seen on 6% SDS-PAGE gel under reducing conditions. The 120-kDa band represents the mature furin-processed minireceptor form that corresponds to the LRP-ligand binding domain 4 (see Fig. 1, and Ref. 30). Since the HA epitope is within the amino terminus of the LRP minireceptors, Western blot analyses with anti-HA antibody do not detect the LRP-85 band. However, the presence of this band was confirmed by metabolic labeling with [35S]cysteine and immunoprecipitation (data not shown). The upper band that migrates ;200 kDa represents the full-length ER precursor form that lacks complex sugar modification (see Ref. 30). mLRP4Ttailess exhibits a similar banding pattern on SDS-PAGE, except that the ER form migrates faster than that FIG. 5. Ligand binding activity and Western blotting analysis of LRP minireceptor expression in LRP-null CHO cells. A, binding of 125I-RAP (5 nM) to LRP-null CHO cells stably transfected with pcDNA3 or various LRP minireceptors was carried out for 1 h at 4 °C in the absence (f) or presence (M) of 500 nM RAP. Values are the average of triple determinations with the S.E. indicated by error bars. B, Western blotting analysis of LRP minireceptor expression in LRP-null CHO cells. Same amounts of cell lysates from LRP-null CHO cells stably transfected with LRP minireceptors were separated via 6% SDS-PAGE under reducing conditions and Western blotted with anti-HA antibody. The positions of the ER forms and the extracellular subunits representing the ligand-binding domain (120 kDa) are labeled. The bottom panel shows densitometric analysis of data described in top panel. Data are calculated as the ratio of 120-kDa/ER form of LRP minireceptors. This experiment is a representative of two such experiments performed with similar data. of mLRP4T100 due to the tail truncation. The results show that, although the ER forms of mLRP4T100 and those mutants defective in endocytosis as well as mLRP4Ttailess are expressed at a similar level, the furin-processed 120-kDa forms are detected at distinctly different levels, i.e. the 120-kDa forms of mLRP4T100(Y63A), mLRP4T100(L66A), mLRP4T100(Y63A,L86A,L87A), and mLRP4Ttailess are significantly increased, while the 120-kDa form of mLRP4T100(L86A,L86A) is only slightly greater than that of mLRP4T100 (Fig. 5B). The ratio of 120-kDa/ER of mLRP4T100 is 0.37, and the ratios of 17192 LRP Endocytosis Signals FIG. 7. Scanning confocal images of LRP minireceptors. LRPnull CHO cells stably transfected with mLRP4T100 (A), mLRP4T100(L86A,L87A) (B), mLRP4T100(Y63A) (C), mLRP4T100(L66A) (D), mLRP4T100(Y63A,L86A,L87A) (E), and mLRP4Ttailess (F) were immunostained with anti-HA antibody and detected with Cy3-Goat antimouse IgG. Images represent single 0.16-mm-thin midsections from scanned cells. FIG. 6. Flow cytometric analysis of cell surface LRP minireceptor expression in LRP-null CHO cells. A, histograms of LRP minireceptor cytofluorimetric analysis in LRP-null CHO cells. LRP-null CHO cells stably transfected with LRP minireceptors were labeled with anti-HA antibody and detected with goat anti-mouse IgFITC. Background fluorescence intensity was assessed in the absence of primary monoclonal antibody (thin line). The x axis represents relative fluorescence intensity, and the y axis represents relative cell number. B, the number of cell surface LRP minireceptors in LRP-null CHO cells. LRPnull CHO cells stably transfected with LRP minireceptors were stained as described above. The number of cell surface minireceptors per cell were determined by using Quantum Simply Cellular microbead standard as described under “Experimental Procedures.” Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of two such experiments performed with similar data. those mutants defective in endocytosis as well as mLRP4Ttailess are ranged from 0.74 to 1.9 (Fig. 5B). Thus, at steady state, mutation of LRP endocytosis signals or deletion of the cytoplasmic tail of LRP results in a significant increase in expression of the mature minireceptors. To address whether the increases in RAP-binding and the expression of the 120-kDa forms are results of cell surface accumulation of the LRP minireceptors, we next measured the cell surface LRP minireceptors by flow cytometric analysis in intact cells. As shown in Fig. 6A, CHO cells transfected with only the pcDNA3 vector yields no specific staining with anti-HA antibody. Wild type mLRP4T100 exhibited a moderate level of cell surface staining. mLRP4T100 mutants defective in endocytosis, however, exhibited dramatic increase in cell surface staining. Using Quantum Simply Cellular bead standards (35), the numbers of LRP minireceptors per cell surface was assessed (Fig. 6B). Higher numbers of LRP minireceptors were found on all mLRP4T100 mutants defective in endocytosis (ranging from ;49,300 to ;388,000 sites/cell), when compared with wild type mLRP4T100 (;36,700 sites/cell), with the high- est numbers observed with mLRP4Ttailess and mLRP4T100(Y63A,L86A,L87A). Taken together, these data clearly demonstrate that mLRP4T100 mutants defective in endocytosis accumulate at the cell surface. To confirm the above results, we determined the steady-state distribution of LRP minireceptors stably transfected in CHO cells via confocal microscopy. As seen in Fig. 7A, wild type mLRP4T100 exhibits both cell surface and vesicular staining, similar to that seen with endogenous LRP (41). Compared with mLRP4T100, the change of cell surface mLRP4T100(L86A,L87A) expression is not significant (Fig. 7B). However, mutations in LRP dominant endocytosis motif YXXL resulted in high levels of expression at the cell surface (Fig. 7, C and D). mLRP4T100(Y63A,L86A,L87A) and mLRP4Ttailess also show markedly increased cell surface localization (Fig. 7, E and F). These observations of increased expression at the cell surface for the various endocytosis mutants support the role for the YXXL motif as a dominant signal in LRP endocytosis. mLRP4T100 Mutants Defective in Endocytosis Exhibit Reduction in the Efficiency of Ligand Degradation—Having established that LRP endocytosis is mediated by the YXXL motif and the distal di-leucine motif, we then investigated the efficiency of LRP ligand degradation for wild type mLRP4T100 and its endocytosis mutants. Fig. 8A shows that the 125I-RAP degradation efficiency of the LRP endocytosis mutants decreased significantly with mLRP4Ttailess showing the lowest level of 125I-RAP degradation. As expected, mLRP4T100(L86A,L87A) shows partially reduced degradation, while mLRP4T100(Y63A), mLRP4T100(L66A), and mLRP4T100(Y63A,L86A,L87A) show only 12.9%, 8.8%, and 6% that of wild type mLRP4T100, respectively. Our recent studies have shown that mLRP4T100 is able to degrade scuPA efficiently.2 Unlike RAP, scuPA is a physiological ligand for LRP. Thus, we utilized scuPA to measure the efficiency of ligand degradation for the LRP minireceptors. Similar to that seen with 125I-RAP, LRP endocytosis mutants exhibited impaired degradation of 125I-scuPA. mLRP4Ttailess showed lowest level of scuPA degradation. mLRP4T100(L86A, 2 L. M. Obermoeller, Y. Li, and G. Bu, unpublished results. 17193 LRP Endocytosis Signals FIG. 8. mLRP4T100 mutants defective in endocytosis exhibit lower ligand degradation efficiency. LRP-null CHO cells stably transfected with mLRP4T100, mLRP4T100(L86A,L87A), mLRP4T100(Y63A), mLRP4T100(L66A), mLRP4T100(Y63A,L86A,L87A), and mLRP4Ttailess were incubated with 5 nM 125I-RAP (A) or 125I-scuPA (B) at 37 °C for 4 h in the presence or absence of 500 nM RAP. The ligand degradation efficiency was determined as described under “Experimental Procedures.” Values are the average of triple determinations with the S.E. indicated by error bars. This experiment is a representative of two such experiments performed with similar data. L87A) showed partially reduced scuPA degradation, while mLRP4T100(Y63A), mLRP4T100(L66A), and mLRP4T100(Y63A,L86A,L87A) exhibited only 30%, 23.1%, and 8.7% that of wild type mLRP4T100, respectively. Taken together, these data clearly demonstrate that cells expressing mLRP4T100 mutants defective in endocytosis exhibit reduced ligand degradation efficiency. DISCUSSION The NPXY motif serves as the signal for rapid endocytosis through coated pits for the LDLR (1, 24). It is the only sequence element within the cytoplasmic tail that is conserved among all members of the LDLR family. The tail of LRP contains two copies of this motif, suggesting that the NPXY sequences might also function as endocytosis signals for LRP. In the present study, we provide direct evidence that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. Substitution of tyrosine or leucine within the YXXL motif with alanine significantly reduced the endocytosis rate. We also found that the distal di-leucine motif contributes to LRP endocytosis, and that its function is independent from the YXXL motif. Additionally, studies of the cell surface expression and efficiency of ligand degradation for LRP endocytosis mutants strongly support these findings. These data suggest that each member of the LDLR family may use different potential endocytosis signal(s) within their cytoplasmic tails. In the present study, we also demonstrated that the proximal NPXY motif and the proximal di-leucine motif function in truncated but not in wild type full-length receptor tails. This suggests that these motifs might be embedded within the wild type receptor tail but exposed in the truncated tails. A similar finding has been reported for the growth hormone receptor (42). Although endocytosis of the wild type growth hormone receptor depends on the ubiquitin system, a truncated form of growth hormone receptor, which lacks the ubiquitin-dependent endocytosis motif, utilizes a di-leucine motif for its endocytosis (42). Alternatively, the function of these potential endocytosis motifs within wild type receptor tail may be overridden by the presence of dominant YXXL motif. Recently, studies have revealed new roles of LDLR family members as transducers of extracellular signals. It has been demonstrated that VLDLR and apoER2 function as obligate components in the Reelin/Disabled-mediated neuronal migration pathway (25–27, 43). A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 is involved in the control of cell position during mammalian brain development (44 – 47). It has been shown that Disabled-1 interacts with the NPXY motifs within the tails of several members of the LDLR family (25, 26). Mice lacking the genes for both VLDLR and apoER2 demonstrate a neurological and neuroanatomical phenotype that is indistinguishable from animals deficient in either Reelin or Disabled-1 (26). Potential signaling functions for members of the LDLR family have also been suggested from other observations. For example, Goretzki and Mueller (48) have shown that the LRP tail interacts with a GTP-binding protein and induces cyclic-AMPdependent protein kinase activity. Similar signal transduction event down stream from LRP was also implicated in hippocampal neurons (49). At present, it is still unclear whether the signaling event(s) initiated by lipoprotein receptors can be regulated by endocytosis. Studies using different experimental systems have revealed that tyrosine-based as well as di-leucine-based sorting signals of membrane proteins can be recognized by adaptor complexes, which in turn associate with clathrin and other accessory molecules to generate clathrin coats and coated transport vesicles (18, 50). Adaptor complex AP-2 plays a critical role in two early steps of the endocytic pathway at the plasma membrane: the formation of the clathrin lattice and the selection of specific cargo proteins for internalization (18, 51). AP-2 interacts with clathrin through its b subunits and promotes coat formation. Interaction of the AP-2 m subunit with receptors containing tyrosine-based as well as di-leucine-based internalization motifs contributes to their localization to coated pits. In the present studies, we have shown that the YXXL motif serves as the dominant motif for LRP endocytosis, and that the distal dileucine motif further contributes to LRP endocytosis. Thus, it will be interesting to examine whether AP-2 plays a major role in LRP endocytosis and whether it interacts directly with the receptor endocytosis signals. In conclusion, our results show that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We have also defined that the distal di-leucine motif contributes to LRP endocytosis, and that its function is independent of the YXXL motif. Mutants defective in endocytosis accumulate at the cell surface, and exhibit reduced efficiency for ligand degradation. These data together with that of other’s, suggest that the YXXL motif and the distal di-leucine motif of the LRP tail serve as endocytosis signals, while the NPXY motifs serve as binding sites for cytosolic signaling proteins. Acknowledgments—We are grateful to Alan Schwartz for critical reading and suggestions on the manuscript. We also thank David FitzGerald (National Institutes of Health, Bethesda, MD) for providing the LRP-null CHO cell line REFERENCES 1. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601– 637 2. Strickland, D. K., Kounnas, M. Z., and Argraves, W. S. (1995) FASEB J. 9, 890 – 898 17194 LRP Endocytosis Signals 3. Yamazaki, H., Bujo, H., Kusunoki, J., Seimiya, K., Kanaki, T., Morisaki, N., Schneider, W. J., and Saito, Y. (1996) J. Biol. Chem. 271, 24761–24768 4. Willnow, T. E., Nykjaer, A., and Herz, J. (1999) Nat. Cell Biol. 1, E157-E162 5. Herz, J., Kowal, R. C., Goldstein, J. L., and Brown, M. S. (1990) EMBO J. 9, 1769 –1776 6. Willnow, T. E., Moehring, J. M., Inocencio, N. M., Moehring, T. J., and Herz, J. (1996) Biochem. J. 313, 71–76 7. Herz,. J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119 – 4127 8. Bu, G., Maksymovitch, E. A., Nerbonne, J. M., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 18521–18528 9. Bu, G. (1998) Curr. Opin. Lipidol. 9, 149 –155 10. Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471–1474 11. Warshawsky, I., Bu, G., and Schwartz, A. L. (1993) J. Clin. Invest. 92, 937–944 12. Weaver, A. M., Hussaini, I. M., Mazar, A., Henkin, J., and Gonias, S. L. (1997) J. Biol. Chem. 272, 14372–14379 13. Webb, D. J., Nguyen, D. H., and Gonias, S. L. (2000) J. Cell Sci. 113, 123–134 14. Holtzman, D. M., Pitas, R. E., Kilbridge, J., Nathan, B., Mahley, R. W., Bu, G., and Schwartz, A. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9480 –9484 15. Kounnas, M. Z., Moir, R. D., Rebeck, G. W., Bush, A. I., Argraves, W. S., Tanzi, R. E., Hyman, B. T., and Strickland, D. K. (1995) Cell 82, 331–340 16. Ulery, P. G., Beers, J., Mikhailenko, I., Tanzi, R. E., Rebeck, G. W., Hyman, B. T., and Strickland, D. K. (2000) J. Biol. Chem. 275, 7410 –7415 17. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993) Annu. Rev. Cell Biol. 9, 129 –161 18. Hirst, J., and Robinson, M. S. (1998) Biochim. Biophys. Acta 1404, 173–193 19. Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 18677–18680 20. Dietrich, J., Hou, X., Wegener, A. M., and Geisler, C. (1994) EMBO J. 13, 2156 –2166 21. Pitcher, C., Honing, S., Fingerhut, A., Bowers, K., and Marsh, M. (1999) Mol. Biol. Cell 10, 677– 691 22. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425– 479 23. Strous, G. J., and Govers, R. (1999) J. Cell Sci. 112, 1417–1423 24. Chen, W. J., Goldstein, J. L., and Brown, M. S. (1990) J. Biol. Chem. 265, 3116 –3123 25. Trommsdorff, M., Borg, J. P., Margolis, B., and Herz, J. (1998) J. Biol. Chem. 273, 33556 –33560 26. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999) Cell 97, 689 –701 27. Hiesberger, T., Trommsdorff, M., Howell, B. W., Goffinet, A., Mumby, M. C., Cooper, J. A., and Herz, J. (1999) Neuron 24, 481– 489 28. Bu, G., Maksymovitch, E. A., and Schwartz, A. L. (1993) J. Biol. Chem. 268, 13002–13009 29. Morton, P. A., Owensby, D. A., Wun, T. C., Billadello, J. J., and Schwartz, A. L. (1990) J. Biol. Chem. 265, 14093–14099 30. Obermoeller, L. M., Chen, Z., Schwartz, A. L., and Bu, G. (1998) J. Biol. Chem. 273, 22374 –22381 31. FitzGerald, D. J., Fryling, C. M., Zdanovsky, A., Saelinger, C. B., Kounnas, M., Winkles, J. A., Strickland, D., and Leppla, S. (1995) J. Cell Biol. 129, 1533–1541 32. Obermoeller, L, M., Warshawsky, I., Wardell, M. R., and Bu, G. (1997) J. Biol. Chem. 272, 10761–10768 33. Bu, G., and Rennke, S. (1996) J. Biol. Chem. 271, 22218 –22224 34. Li, Y., Wood, N., Yellowlees, D., and Donnelly, P. (1997) Biochem. Biophys. Res. Commun. 240, 122–127 35. Zagursky, R. J., Sharp, D., Solomon, K. A., and Schwartz, A. (1995) BioTechniques 18, 504 –509 36. Bu, G., Morton, P. A., and Schwartz, A. L. (1992) J. Biol. Chem. 267, 15595–15602 37. Vilhardt, F., Nielsen, M., Sandvig, K., and van Deurs, B. (1999) Mol. Biol. Cell 10, 179 –195 38. York, S. J., Arneson, L. S., Gregory, W. T., Dahms, N. M., and Kornfeld, S. (1999) J. Biol. Chem. 274, 1164 –1171 39. Lai, A., Sisodia, S. S., and Trowbridge, I. S. (1995) J. Biol. Chem. 270, 3565–3573 40. Vassiliou, G., and Stanley, K. K. (1994) J. Biol. Chem. 269, 15172–15178 41. Bu, G., Maksymovitch, E. A., Geuze, H., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 29874 –29882 42. Govers, R., van Kerkhof, P., Schwartz, A. L., and Strous, G. J. (1998) J. Biol. Chem. 273, 16426 –16433 43. D’Arcangelo, G., Homayouni, R., Keshvara, L., Rice, D. S., Sheldon, M., and Curran, T. (1999) Neuron 24, 471– 479 44. Howell, B. W., Hawkes, R., Soriano, P., and Cooper, J. A. (1997) Nature 389, 733–737 45. Sheldon, M., Rice, D. S., D’Arcangelo, G., Yoneshima, H., Nakajima, K., Mikoshiba, K, Howell, B. W., Cooper, J. A., Goldowitz, D., and Curran, T. (1997) Nature 389, 730 –733 46. Ware, M. L., Fox, J. W., Gonzalez, J. L., Davis, N. M., Lambert de Rouvroit, C., Russo, C. J., Chua, S. C. Jr., Goffinet, A. M., and Walsh, C. A. (1997) Neuron 19, 239 –249 47. Curran, T., and D’Arcangelo, G. (1998) Brain Res. Brain Res. Rev. 26, 285–294 48. Goretzki, L., and Mueller, B. M. (1998) Biochem. J. 336, 381–386 49. Zhuo, M., Holtzman D. M., Li, Y., Osaka, H., DeMaro, J., Jacquin, M., and Bu, G. (2000) J. Neurosci. 20, 542–549 50. Dell’Angelica, E. C., Mullins, C., and Bonifacino, J. S. (1999) J. Biol. Chem. 274, 7278 –7285 51. Bonifacino, J. S., and Dell’Angelica, E. C. (1999) J. Cell Biol. 145, 923–926