Identification of an apical sorting determinant in the cytoplasmic tail of megalin

Tetsuro Takeda1, Hajime Yamazaki1, and Marilyn G. Farquhar1,2

Departments of 1 Cellular and Molecular Medicine and 2 Pathology, University of California, San Diego, La Jolla, California 92093


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Megalin is the main endocytic receptor of the proximal tubule and is responsible for reabsorption of many filtered proteins. In contrast to other members of the low-density lipoprotein (LDL) receptor gene family, it is expressed on the apical plasma membrane (PM) of polarized epithelial cells. To identify megalin's apical sorting signal, we generated deletion mutants and chimeric minireceptors composed of complementary regions of megalin and LDL receptor-related protein (LRP) and assessed the distribution of the mutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescence and cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, of MDCK cells. We found that the information that directs apical sorting is present in the cytoplasmic tail (CT) of megalin, which contains three NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-binding motif at its COOH terminus. Deletion analysis established that amino acids 107-136 of the megalin-CT containing the second NPXY-like motif are critical for apical sorting and targeting, whereas the regions containing the first and third NPXY motifs are required for efficient endocytosis. We conclude that the megalin-CT contains a novel apical sorting determinant and that cytoplasmic sorting machinery exists in MDCK cells for some apical transmembrane proteins.

Madin-Darby canine kidney cells; low-density lipoprotein receptor-related protein; chimeric minireceptors; NPXY sorting signals; endocytosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLARIZED EPITHELIAL CELLS vectorially transport ions and solutes between the outside environment and internal milieu to maintain ionic homeostasis. A key feature of such cells is that they possess apical and basolateral plasma membrane (PM) domains of distinct lipid and protein composition (39) with distinct sorting determinants (19, 21). The basolateral sorting signals identified to date are short motifs located in the cytoplasmic tail (CT) of transmembrane (TM) proteins, of which the best characterized are the tyrosine (NPXY or YXXØ) (11, 39) and dileucine (18) motifs that bind to clathrin adaptor protein complexes AP-1 and AP-2 (22, 28). Recently a novel adaptor AP-1B was discovered that is directly involved in sorting a class of basolateral TM proteins (8). Apical sorting signals are less well defined, but they can reside in either the luminal domain or membrane anchor of the targeted protein and have been proposed to be present in protein, carbohydrate, or lipid moieties (29). Megalin is an endocytic receptor expressed in clathrin-coated pits at the apical PM of a number of epithelia, including those of the kidney proximal tubule (5, 14). Megalin binds and internalizes multiple ligands from the glomerular filtrate, including albumin, peptide hormones, and vitamin carriers (3, 25). Megalin is a member of the LDL receptor gene family (30) and is closely related to the LDL receptor-related protein (LRP) (15). In contrast to megalin, LRP is expressed on the basolateral PM domain in epithelial cells (e.g., hepatocytes) (40). Thus, although megalin and LRP bind to many of the same ligands and their domain organization is similar, their targeting in polarized epithelial cells is distinct. How the cellular machinery sorts these two related receptors for delivery to the appropriate PM domains has not been determined.

In this study, we generated deletion mutants and chimeras of megalin and LRP minireceptors and expressed them in Madin-Darby canine kidney (MDCK) cells to identify the regions that specify polarized targeting. We found that these receptors possess distinct sorting information in their CTs, and we have pinpointed the unique region in the megalin-CT responsible for apical sorting.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Rat megalin partial cDNA (clone 217) was obtained previously (30), and human full-length LRP cDNA was provided by Dr. Joachim Herz (Univ. of Texas Southwestern Medical Center, Dallas, TX). Enhanced chemiluminescent substrate (SuperSignal) was obtained from Pierce (Rockford, IL). Primers were obtained from Life Technologies (Gaithersburg, MD), and restriction enzymes were from New England Biolabs (Beverly, MA). Human lactoferrin was purchased from CalBiochem (San Diego, CA), and [35S]EasyTag Express protein-labeling mix (~1,000 Ci/mmol) was from DuPont NEN. Transwell filters (12 mm, 0.4-µm pore size) were obtained from Costar. Chemical reagents were from Sigma except as indicated. Receptor-associated protein (RAP)-glutathione-S-transferase (GST) fusion protein was prepared as previously described (24).

Antibodies. Rabbit antibodies raised against the fourth ligand binding domain (LBD) of megalin (38) were previously described. Monoclonal anti-HA (12CA5) was obtained from CRP (Richmond, CA). Highly cross-adsorbed Alexa 488-conjugated goat anti-rabbit and Alexa 594-conjugated goat anti-mouse F(ab')2 were purchased from Molecular Probes (Eugene, OR). Affinity-purified goat anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase (HRP) were from Bio-Rad Laboratories (Hercules, CA).

cDNA constructs. Rat RAP cDNA (24) was digested with SmaI and EcoRV. The released fragment was subcloned into pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA), designated pcDNA/Zeo-RAP. To generate megalin minireceptor, a PCR fragment encompassing the fourth ligand-binding repeat through the CT (nucleotides 10515-14129, amino acids 3440-4635) (Fig. 1) (30) was cloned into the pLNCX2 retroviral vector (ClonTech, Palo Alto, CA). The resulting plasmid, designated pLNCX-M4, was used as the base plasmid for the construction of cDNAs for mutagenesis. To generate a hemagglutinin (HA)-tagged construct, pSPH-HA, we inserted the sequence encoding the HA epitope (YPYDVPDYA) immediately downstream of the signal peptide cleavage site of the pSPH vector (31) by using an Exsite Mutagenesis kit (Stratagene, La Jolla, CA). To generate LRP minireceptor, a PCR fragment encompassing the fourth LBD through the CT of LRP (nucleotides 10343-14101, amino acids 3274-4544) (Fig. 1) (12) was cloned into pLNCX2 (pLNCX-L4). Megalin/LRP chimeric minireceptors were constructed using a Seamless Cloning kit (Stratagene). Primers introducing an Eam1104I site were employed to amplify both pLNCX-M4 and LRP cDNA. The resulting PCR products were digested with Eam1104I and ligated. Two such chimeric receptors were prepared: M4/Lct, containing the fourth LBD and TM domain of megalin and the LRP-CT, and L4/Mct, containing the fourth LBD and TM domain of LRP and megalin-CT.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Megalin and LDL receptor-related protein (LRP) minireceptors. Megalin and LRP contain four ligand-binding domains (LBD1-4). Megalin minireceptor M4 includes LBD4 and the transmembrane (TM) domain and cytoplasmic tail (CT) of megalin. LRP minireceptor L4 includes LBD4 and the TM domains and CT of LRP. The chimeric receptor M4/Lct contains LBD4 and the TM domains of megalin fused to the LRP-CT. The L4/Mct chimera contains LBD4 and the TM domains of LRP fused to megalin-CT. A hemagglutinin (HA) epitope was inserted into L4 and L4/Mct immediately after the signal peptide cleavage site at the NH2 terminus.

To generate sequential deletion mutants of the megalin-CT, we used an Exsite Mutagenesis kit. 5'-Phosphorylated primers were employed to amplify deletion mutants with pLNCX-M4 as a template. The resulting PCR products were self-ligated. The affected region was sequenced to verify the accuracy of PCR products by automated sequencing. PCR primer sequences are available upon request.

Cell culture and generation of MDCK-RAP cells. MDCK and HEK293T cells were maintained in MEM Earles or DMEM Hi-glucose, respectively, supplemented with 10% FCS and 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate (Life Technologies) at 37°C and 5% CO2. To make a cell line stably overexpressing RAP, pcDNA/Zeo-RAP was transfected into MDCK cells using Lipofectamine (Life Technologies) as described previously (36), and transfectants were selected with 0.25 mg/ml zeocin (Invitrogen). As shown by immunoblotting, MDCK-RAP cells expressed approximately five times as much RAP as wild-type cells (data not shown).

Retrovirus production and transfer. High-titer, helper-free retrovirus was prepared by transient transfection using the kat ecotropic packaging system (6). Briefly, HEK293T cells were transfected with a retroviral construct together with a pKat2 packaging construct (provided by Dr. Mark Kamps, Univ. of California, San Diego) using calcium phosphate. Supernatants were collected 48 h posttransfection, passed through a 0.45-µm filter, and frozen at -70°C. MDCK-RAP cells were infected with retrovirus in the presence of 8 µg/ml polybrene (Sigma-Aldrich), and cells were selected in 0.8 mg/ml genetecin (Life Technologies) 48 h postinfection.

Immunofluorescence. MDCK monolayers were fixed with 2% paraformaldehyde in 0.2 M phosphate buffer, pH 7.4 (45 min), permeabilized with 0.1% Triton X-100 in PBS (10 min), and incubated sequentially with anti-megalin LBD4 (1:400) or anti-HA (12CA5) (1:200) and appropriate secondary antibodies (1:250) as described previously (36). Cells were examined with a Bio-Rad confocal microscope (MRC 1024) equipped with Lasersharp 3.1 software (Bio-Rad) and a krypton-argon laser. Images were processed with Adobe Photoshop version 5 software (Adobe Systems, San Jose, CA).

SDS-PAGE and immunoblotting. Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce). Samples were separated on 6 or 7.5% SDS-PAGE under reducing conditions, transferred to polyvinylidene difluoride membranes, and incubated for 2 h at room temperature with primary antibodies, followed by HRP-conjugated goat anti-rabbit IgG (1 h, 1:3,000 dilution) and detection by enhanced chemiluminescence (ECL).

Domain-selective biotinylation assay. Cell surface biotinylation was performed essentially as described previously (32). Briefly, 2 × 105 MDCK cells were seeded on Transwell filters and grown for 5 days, after which the surface proteins on either the apical or basal side of the membranes were biotinylated by exposure to 0.5 ml of NHS-ss-biotin (Pierce) in PBS/Ca2+/Mg2+ for 20 min at 4°C. After quenching, filters were excised and cells were lysed with 0.5 ml lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 7.5, and 0.2% BSA) containing protease inhibitors (1 mM PMSF and 10 µg/ml leupeptin) for 1 h at 4°C. Biotinylated proteins were recovered on streptavidin-agarose beads (Pierce). Bound proteins were eluted by incubating the beads in Laemmli sample buffer at 95°C for 5 min, separated by SDS-PAGE, and analyzed by immunoblotting. Quantification of protein bands was done by densitometry with ScanAnalysis software (Alltech Associates, Deerfield, IL), and the percentage of the receptor present on the apical and basolateral cell surfaces was determined.

Biosynthetic transport of receptors to the surface. MDCK cells (1 × 106) grown for 5 days on Transwell filters were incubated for 20 min in methionine- and cysteine-free DMEM, pulse-labeled with [35S]EasyTag Express protein-labeling mix from the basolateral side for 20 min, and chased for 0.5, 1, 2, and 4 h in medium containing excess unlabeled methionine and cysteine (16). At the end of each chase, cells were biotinylated from the apical or basolateral surface at 4°C and lysed, and then megalin and LRP minireceptors were immunoprecipitated by incubation overnight with anti-megalin LBD4 or anti-HA, followed by collection on protein A-Sepharose (2 h). Immunoprecipitated proteins were eluted by boiling in 10% SDS and diluted 50-fold with lysis buffer, and biotinylated proteins were recovered by incubation with streptavidin-agarose. Proteins were separated by SDS-PAGE and exposed to BioMax MR X-ray film (Eastman Kodak, Rochester, NY) for 4 days.

Radioiodination. RAP-GST and lactoferrin were radiolabeled using Na-125I (DuPont NEN) and IODO-BEADs (Pierce) according to the manufacturer's instructions. Specific activities were as follows: 125I- RAP-GST, ~4,000 cpm/ng; 125I-lactoferrin, ~6,000 cpm/ng.

Internalization and degradation of 125I-labeled ligands. MDCK-RAP cells (2 × 105) grown for 5 days on Transwell filters were incubated with 125I-labeled ligands (5 nM) for 2 h at 37°C in the presence or absence of nonradiolabeled ligand (100 nM). Medium was then removed, adjusted to 15% trichloroacetic acid (TCA) at 4°C, and centrifuged (15,000 g for 30 min), and TCA-soluble counts [indicating cellular degradation of 125I-labeled ligands (4)] were quantified by gamma counting. To correct for liberation of iodine from 125I-labeled ligands, TCA-soluble radioactivity in medium incubated without cells was subtracted from that found in the samples. The degradation of 125I-labeled ligands was normalized to total cell protein.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Targeting of megalin and LRP minireceptors in MDCK cells. The very large size of megalin (~600 kDa) and LRP (~515 kDa) limits molecular manipulations at the cDNA level and the expression of these proteins via transfection. Thus we generated megalin (M4) and LRP (L4) minireceptors that mimic the function and trafficking of these receptors (12) (Fig. 1). An HA epitope was included near the NH2 terminus of LRP constructs for immunodetection. MDCK cells do not express endogenous megalin, but they do express endogenous LRP. We prepared MDCK-RAP cells that stably overexpress five times as much RAP as wild-type MDCK cells (data not shown) to facilitate delivery of minireceptors to the cell surface. RAP serves as a specialized chaperone for LDL receptor family members (2).

To increase the number of MDCK cells expressing megalin and LRP, we used the kat retrovirus system (6). When filter-grown cells were stained with anti-megalin LBD4 or anti-HA (for LRP detection) and examined by confocal microscopy, megalin minireceptor M4 was distributed only on the apical surface (Fig. 2, a and b), whereas LRP minireceptor L4 was expressed only on the basolateral surface (Fig. 2, c and d) as seen in both en face and vertical (X-Z) views. Thus, in MDCK cells, megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, which is the typical location of endogenous receptors. Because the viral-infected, genetecin-selected MDCK cell lines expressing the receptors were not clonal, variations in the level of expression of the receptors were seen among cells in a given field.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2.   Immunofluorescence staining of megalin, LRP, and chimeric minireceptors stably expressed in MDCK-RAP (receptor-associated protein) cells. a and b: M4 megalin minireceptor is correctly targeted to the apical surface above the level of the tight junction as shown in both en face (top) and X-Z vertical sections (bottom). c and d: L4 LRP minireceptor is correctly targeted to the basolateral surface. e and f: M4/Lct chimeras. When the LRP-CT is exchanged for the megalin-CT, megalin is redirected and targeted basolaterally. g and h: L4/Mct chimeras. When the megalin-CT is substituted for that of LRP, LRP is redirected to the apical surface. Filter-grown cells were immunostained with anti-megalin LBD4 (a, b, e, and f) or anti-HA (c, d, g, and h) for LRP. X-Z vertical sections were taken in 0.1-µm steps through the cells. Bars, 10 µm.

The CTs of megalin and LRP contain sorting determinants. Because the ectodomains of megalin and LRP are highly homologous and their cytoplasmic domains have no similarity except for the presence of NPXY motifs (31), we reasoned that the sorting determinants might reside in their cytoplasmic domains. To find out whether this is the case, we swapped their CTs and generated two chimeric receptors, M4/Lct and L4/Mct (see Fig. 1), infected MDCK-RAP cells with the retrovirus constructs, and analyzed their sorting behavior. The M4/Lct minireceptor, containing the fourth LBD and TM domain of megalin and the LRP-CT, was predominantly expressed on the basolateral PM (Fig. 2, e and f), whereas L4/Mct, containing the fourth LBD and TM domain of LRP and megalin-CT, was expressed on the apical PM (Fig. 2, g and h). These results indicate that the CTs of megalin and LRP contain the information necessary for polarized sorting and targeting.

To confirm and extend the immunocytochemical results, we quantitated the distribution of the receptors on the apical and basolateral surfaces of MDCK cells by using a domain-selective biotinylation assay (32) whereby cells grown on filters are biotinylated from either the apical or basal side, and the biotinylated cell surface proteins are selectively collected on streptavidin-agarose (Fig. 3A). Most (~93%) of the M4 was detected as an ~200-kDa band on the apical PM when the cells were biotinylated at the apical side, whereas most (~92%) of the M4/Lct chimera was detected on the basolateral PM. By contrast, L4 was mainly (~99%) on the basolateral PM, and the L4/Mct chimera was found predominantly (98%) at the apical PM. These results validate that the CTs contain the information necessary for selective targeting of megalin and LRP to the apical and basolateral domains of the PM.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 3.   Targeting of megalin, LRP, and chimeric receptors. A: surface biotinylation. M4 (lanes 1 and 2) and L4/Mct (lanes 7 and 8) are present mainly (>90%) on the apical (A) surface, whereas L4 (lanes 5 and 6) and M4/Lct (lanes 3 and 4) are present mainly (>90%) on the basolateral (B) surface. Quantification of the protein bands is shown by graph. MDCK cells expressing the indicated minireceptors were grown on Transwell filters and biotinylated on either the apical or basal side of the filter. Biotinylated proteins were recovered from cell lysates on streptavidin-agarose beads, separated by 6% SDS-PAGE, transferred to membranes, and blotted with anti-megalin LBD4 or anti-HA. The percentage of the biotinylated receptors found on the apical and basolateral cell surfaces was determined by densitometry. Data represent means ± SD of 3 separate experiments. B: biosynthetic studies. At the end of a 20-min pulse (0 h), newly synthesized M4 (top) is already detected at the apical surface of MDCK cells. At 0.5 h of chase, the amount of newly synthesized, labeled receptor increases and continues to increase up to 2 h, when ~90% are found on the apical surface. L4 (~200 kDa; bottom) begins to appear at the cell surface at the end of a 20-min pulse (0 min) and is found largely basolaterally. After 0.5 h of chase, the proteolytically processed forms of the receptor (~85 and ~120 kDa) are seen, and the amount of surface receptor continues to increase up to 2 h. The minireceptor includes the furin cleavage site (see Fig. 1) and is processed normally. MDCK cells expressing M4 and L4 minireceptors cultured on Transwell filters were pulse-labeled with [5S]EasyTag for 20 min and chased for the times indicated. After each chase period, cells were biotinylated from either the apical or basal side of the filter, and immunoprecipitation was carried out on cell lysates with anti-megalin LBD4 or anti-HA. Precipitated proteins were eluted, bound to streptavidin-agarose, and separated by SDS-PAGE, and then radiolabeled, biotinylated receptor was visualized by autoradiography.

Megalin and LRP are directly targeted to the apical and basolateral cell surfaces. To investigate the intracellular routing of megalin and LRP minireceptors in MDCK cells, we traced the cell surface delivery of newly synthesized receptors by using a pulse-chase/membrane-targeting assay (16). Filter-grown cells were pulse-labeled (20 min), chased (up to 4 h), and biotinylated from either the apical or basolateral surface at the end of each chase period, and then immunoprecipitation was carried out with anti-megalin LBD4 or anti-HA. The precipitated protein was subsequently absorbed on streptavidin-agarose to selectively recover only the radiolabeled receptors that had reached the cell surface at the time of biotinylation. We found that newly synthesized M4 was detected on the apical surface throughout the chase period with 80% of the total on the apical surface at 0.5 and 1 h of chase and 90% at 2 and 4 h (Fig. 3B, top), whereas LRP minireceptor L4 was predominantly targeted to the basolateral side at all chase times tested (Fig. 3B, bottom). These results suggest that, as is the case with other membrane proteins, these receptors follow a direct route from the trans-Golgi network to the apical or basolateral PM in MDCK-RAP cells.

Residues 107-136 in the megalin-CT are necessary for apical sorting. The megalin-CT contains a number of intriguing motifs, including NPXY, NPXY-like, YXXØ, dileucine, SH3, and PKC motifs and a COOH-terminal PDZ-binding motif (Fig. 4). To identify which of these motifs is necessary for apical sorting, we constructed COOH-terminal deletion mutants with sequential truncations of the CT (Fig. 4). When the truncated receptors were stably expressed in MDCK-RAP cells, M4ct210, M4ct170, and M4ct136 were correctly targeted to the apical domain (Fig. 5, a-f), whereas M4ct106, M4ct69, M4ct34, and M4ct1 were expressed on both the apical and basolateral PM (Fig. 5, g-n). Quantification of the proportion of truncated receptors targeted to each membrane domain, using the domain-selective biotinylation assay, demonstrated that >90% of M4ct210, M4ct170, and M4ct136 were present at the apical surface (Fig. 6, lanes 3-8). The constructs with the further truncated receptors, M4ct210, M4ct69, Mct34, and M4ct1, were expressed on both the apical and basolateral cell surfaces at similar levels (Fig. 6, lanes 9-16). We conclude that 1) the region of the CT located between residues 107 and 136 containing the NPXY-like motif and the first YXXØ motif contains the information required for apical sorting, and 2) the deleted third NPXY and second YXXØ motifs, SH3 recognition site, and PDZ binding motif are not required for sorting and apical targeting of megalin.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Schematic representation of megalin-CT and truncation mutants showing potential endocytosis motifs and apical sorting signals. The megalin-CT contains 2 NPXY motifs, an NPXY-like motif, 2 YXXO motifs, and 1 each of dileucine, SH3, PKC, and PDZ-binding motifs. The location of the truncated receptors on the apical and basolateral domains in MDCK-RAP cells (based on data in Figs. 5 and 6) and the endocytosis efficiency relative to megalin minireceptor M4 with an intact CT (set at 100%; based on data in Fig. 8) is also indicated.



View larger version (99K):
[in this window]
[in a new window]
 
Fig. 5.   Immunofluorescence localization of COOH-terminal truncated megalin minireceptors expressed in MDCK-RAP cells. M4ct210 (a and b) with the PDZ-binding motif deleted from the COOH terminus of the CT, M4ct170 (c and d) with the PDZ-binding motif and the SH3 domain deleted, and M4ct136 (e and f) with the third NPXY motif also deleted are expressed mainly on the apical surface of MDCK cells as shown in both en face (top) and X-Z vertical sections (bottom). The shorter mutants M4ct106 (g and h) with the NPXY-like domain deleted, as well as M4ct69 (i and j), M4ct34 (k and l), and M4ct1 (m and n) are localized on both apical and basolateral surfaces. Filter-grown cells were immunostained with anti-megalin LBD4 as described in Fig. 2. Bars, 10 µm.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 6.   Targeting of truncated megalin minireceptors to the apical or basolateral PM of MDCK cells. M4 (lanes 1 and 2), as well as the M4ct210 (lanes 3 and 4), M4ct170 (lanes 5 and 6), and M4ct136 (lanes 7 and 8) deletion mutants, is present mainly (>90%) at the apical surface, whereas the shorter mutants M4ct106 (lanes 9 and 10), M4ct69 (lanes 11 and 12), M4tct34 (lanes 12 and 13), and M4ct1 (lanes 14 and 15) are randomly distributed on both the apical and basolateral cell surfaces at similar levels. Cells expressing the indicated truncated megalin minireceptors were biotinylated from either the apical or basal surface, and biotinylated proteins were prepared as described in Fig. 3A. Top: immunoblotting of biotinylated proteins with anti-LBD4. Bottom: densitometric quantification of the protein bands at top presented as percentages of total (apical and basolateral) biotinylated cell surface receptors. Data represent means ± SD of 3 separate experiments.

Selective internalization of ligands from the apical or basolateral cell surface. To assess the functionality of the receptors expressed in MDCK cells, we determined the ability of the cells to take up and degrade 125I-lactoferrin (a ligand for both megalin and LRP) from either the apical or basal side of the Transwell chamber by quantifying the TCA-soluble radioactivity released into the medium. When 125I-lactoferrin was added to the apical chamber (Fig. 7A), M4 cells or L4/Mct cells expressing the LRP minireceptor chimera with the megalin-CT rapidly took up and degraded lactoferrin, whereas wild-type cells, L4 cells, or M4/Lct cells expressing megalin minireceptor chimeras with the LRP-CT failed to take up and degrade the ligand. Uptake by cells expressing constructs containing megalin-CT was 20- to 30-fold more efficient than that of wild-type cells and those expressing constructs containing the LRP-CT. When 125I-lactoferrin was added to the basal chamber (Fig. 7B), L4 minireceptors and the M4/Lct chimera containing the LRP-CT showed greater degradation (~1.5- to ~2.5-fold) than that of wild-type and other mutant cell lines. Similar results were obtained when RAP was used in the same ligand degradation assay (data not shown). The higher base uptake of 125I-lactoferrin added to the basal surface is due to the expression of endogenous LRP. Thus most of the functional M4 and L4/Mct receptors are localized at the apical domain of infected MDCK cells, whereas the functional L4 and M4/Lct receptors are predominantly found at the basolateral PM.


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 7.   Degradation of 125I-labeled lactoferrin by megalin, LRP, and chimeric minireceptors. A: after lactoferrin was added to the apical chamber, cells expressing M4 and L4/Mct efficiently took up and degraded the ligand, but wild-type (wt) MDCK-RAP cells, as well as cells expressing L4 and M4/Lct, degraded little lactoferrin. B: when lactoferrin was added to the basal chamber, cells expressing L4 and M4/Lct degraded lactoferrin more efficiently (~1.5- to 2.5-fold) than wt cells and other mutant cell lines. 125I-lactoferrin was added to the apical or basal chamber of filter-grown MDCK cells expressing chimeric minireceptors. Cells were then incubated at 37°C for 2 h, after which medium was precipitated with 15% trichloroacetic acid (TCA). TCA-soluble counts (indicating ligand degradation) were determined and expressed as cpm/mg total cell protein. Data represent means ± SE of 3 separate experiments performed in duplicate.

The apical sorting signal of megalin differs from its endocytosis signal. The single NPXY motif of the LDL receptor is known to serve as the signal for receptor-mediated endocytosis via coated pits (9). The megalin-CT contains two NPXY motifs and a NPXY-like motif. To establish which of these contribute to rapid endocytosis, we compared ligand uptake and degradation in cells expressing megalin minireceptors with sequential truncation of their CT. When 125I-lactoferrin was added apically (Fig. 8), cells expressing M4ct210 and M4ct170 internalized and degraded lactoferrin as efficiently as cells expressing M4 receptor with an intact CT. However, cells expressing M4ct136 and M4ct106 showed decreased (~50%) degradation of lactoferrin, and cells expressing M4ct69, lacking all three NPXY motifs, were least efficient (~20%). The fact that M4ct106 cells with both the second and third NPXY motifs deleted were as efficient as M4ct136 cells lacking only the third NPXY motif indicates that the second NPXY motif is not essential for endocytosis. Collectively, our results suggest that regions containing the first and third NPXY motifs are required for efficient ligand internalization, whereas the second NPXY-like motif is necessary for apical sorting. Thus the apical sorting signal of megalin differs from its endocytosis signal.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   Degradation of 125I-lactoferrin by truncated megalin minireceptors. Cells expressing M4ct210 and M4ct170 internalized and degraded 125I-lactoferrin as efficiently as cells expressing M4 minireceptors with an intact CT. Cells expressing shorter receptors M4ct136, M4ct106, and M4ct69 were less efficient in degradation of lactoferrin (~50, ~55, and ~20%, respectively). 125I-lactoferrin was added to the apical chamber of filter-grown MDCK cells expressing truncated megalin receptors and incubated for 2 h at 37°C, and then TCA-soluble radioactivity was determined as described in Fig. 7.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In polarized MDCK epithelial cells, apical and basolateral TM proteins are sorted into distinct carrier vesicles that bud from the trans-Golgi network and carry these proteins directly to the apical or basolateral PM (13, 17, 19, 21). Thus these proteins contain sorting information that specifies their destination. We have demonstrated that the endocytic receptors megalin and LRP, which have similar functions but are located on different PM domains, contain information in their COOH-terminal CTs that is required for polarized sorting. Megalin minireceptor M4 is correctly targeted to the apical cell surface in MDCK cells, whereas the highly homologous LRP minireceptor L4 is targeted to the basolateral PM under the same conditions. The presence of a sorting determinant in the CT of these receptors was demonstrated by domain-swapping experiments showing that the CT was sufficient to redirect targeting: when the LRP-CT was replaced with that of megalin, the resulting chimera (L4/Mct) was targeted to the apical surface, whereas the corresponding M4/Lct chimera was exclusively delivered to the basolateral surface. Moreover, megalin constructs lacking the entire CT were not sorted and were detected at both surfaces. These results demonstrate the existence of a unique apical targeting signal in the megalin-CT.

A number of molecular signals appear to be involved in establishing the polarized distribution of TM proteins and in regulating their functional tenure at the PM. Basolateral sorting signals identified to date such as NPXY, YXXØ, and dileucine motifs are all located in the CTs of TM proteins and selectively bind to clathrin adaptor protein complexes AP-1, AP-2 (22), or AP-1B (7, 8). In contrast, there are multiple types of apical sorting signals, including glycosylphosphatidylinositol (GPI) anchors (1) and glycans (33), among others (29). One major model for apical sorting is the formation of rafts, composed of glycosphingolipids, cholesterol, GPI-anchored proteins, and certain other proteins clustered in the exoplasmic leaflet of the Golgi and PM (35). However, apical transport of many membrane proteins occurs independently of rafts. COOH-terminal PDZ-binding motifs have also been shown to be capable of mediating apical sorting of the cystic fibrosis transmembrane conductance regulator (CFTR) (34) and the gamma -aminobutyric acid transporter GAT-3 (20). The megalin-CT also contains a PDZ-binding motif (SDV), but deletion of the last three COOH-terminal amino acids had no effect on the apical localization of megalin. Analysis of sequential deletion mutants of the megalin-CT with the use of domain-selective biotinylation and functional assays revealed that the middle region of megalin-CT (amino acids 107-136), containing the second NPXY-like and first YXXØ motifs, is responsible for apical sorting. This suggests that megalin may use a targeting mechanism different from that of other apical TM proteins.

Recently, we (27) and others (10, 23, 26) have demonstrated that several cytoplasmic adaptor and scaffold proteins, including ANKRA, MAGI-1, Dab1, Dab2, JIP-1, JIP-2, PSD-95, CAPON, and GIPC, bind to the megalin-CT. The LDL receptor and LRP interact with the neuronal adaptor proteins Dab1 and FE65 through their NPXY motifs located in their CT (37). Similarly, megalin was recently shown to bind to Dab2 through one of the NPXY motifs in its CT (23). The NPXY motif was the first endocytosis signal discovered (9), and it is well-known to serve as the sorting signal for rapid endocytosis of the LDL receptor via clathrin-coated pits (9, 15). It also overlaps with the basolateral sorting signal for this receptor (11, 39). This led us to investigate whether one or more of the NPXY motifs in the megalin-CT also function as endocytosis signals. Using ligand degradation assays, we found that the regions of the megalin CT containing the first and third NPXY motifs are required for efficient endocytosis.

It remains to be determined how sorting signals are interpreted and with what molecular machinery they interact. Defining the protein(s) that binds to these signals should provide valuable clues to the nature of the molecular mechanisms used to establish and modify the polarized distributions of these physiologically important endocytic receptors in epithelial cells.


    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grant DK-17724.


    FOOTNOTES

Present address of T. Takeda: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.

Present address of H. Yamazaki: Kidney Center, Nagaoka Red Cross Hospital, Nagaoka 940-2085, Japan.

Address for reprint requests and other correspondence: M. G. Farquhar, Dept. of Cellular and Molecular Medicine, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651 (E-mail: mfarquhar{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 8, 2003;10.1152/ajpcell.00514.2002

Received 6 November 2002; accepted in final form 13 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Brown, DA, and Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68: 533-544, 1992[ISI][Medline].

2.   Bu, G, and Rennke S. Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein. J Biol Chem 271: 22218-22224, 1996[Abstract/Free Full Text].

3.   Christensen, EI, and Willnow TE. Essential role of megalin in renal proximal tubule for vitamin homeostasis. J Am Soc Nephrol 10: 2224-2236, 1999[Free Full Text].

4.   Czekay, RP, Orlando RA, Woodward L, Lundstrom M, and Farquhar MG. Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes. Mol Biol Cell 8: 517-532, 1997[Abstract].

5.   Farquhar, MG, Kerjaschki D, Lundstrom M, and Orlando RA. gp330 and RAP: the Heymann nephritis antigenic complex. Ann NY Acad Sci 737: 96-113, 1994[Abstract].

6.   Finer, MH, Dull TJ, Qin L, Farson D, and Roberts MR. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 83: 43-50, 1994[Abstract/Free Full Text].

7.   Folsch, H, Ohno H, Bonifacino JS, and Mellman I. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99: 189-198, 1999[ISI][Medline].

8.   Folsch, H, Pypaert M, Schu P, and Mellman I. Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. J Cell Biol 152: 595-606, 2001[Abstract/Free Full Text].

9.   Goldstein, JL, Brown MS, Anderson RG, Russell DW, and Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol 1: 1-39, 1985[ISI].

10.   Gotthardt, M, Trommsdorff M, Nevitt MF, Shelton J, Richardson JA, Stockinger W, Nimpf J, and Herz J. Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction. J Biol Chem 275: 25616-25624, 2000[Abstract/Free Full Text].

11.   Heilker, R, Spiess M, and Crottet P. Recognition of sorting signals by clathrin adaptors. Bioessays 21: 558-567, 1999[ISI][Medline].

12.   Herz, J, Hamann U, Rogne S, Myklebost O, Gausepohl H, and Stanley KK. Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J 7: 4119-4127, 1988[Abstract].

13.   Ikonen, E, and Simons K. Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin Cell Dev Biol 9: 503-509, 1998[ISI][Medline].

14.   Kerjaschki, D, and Farquhar MG. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc Natl Acad Sci USA 79: 5557-5581, 1982[Abstract].

15.   Krieger, M, and Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 63: 601-637, 1994[ISI][Medline].

16.   Le Bivic, A, Sambuy Y, Mostov K, and Rodriguez-Boulan E. Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells. J Cell Biol 110: 1533-1539, 1990[Abstract].

17.   Matter, K. Epithelial polarity: sorting out the sorters. Curr Biol 10: R39-R42, 2000[ISI][Medline].

18.   Matter, K, Yamamoto EM, and Mellman I. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J Cell Biol 126: 991-1004, 1994[Abstract].

19.   Mostov, KE, Verges M, and Altschuler Y. Membrane traffic in polarized epithelial cells. Curr Opin Cell Biol 12: 483-490, 2000[ISI][Medline].

20.   Muth, TR, Ahn J, and Caplan MJ. Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3. J Biol Chem 273: 25616-25627, 1998[Abstract/Free Full Text].

21.   Nelson, WJ, and Yeaman C. Protein trafficking in the exocytic pathway of polarized epithelial cells. Trends Cell Biol 11: 483-486, 2001[ISI][Medline].

22.   Ohno, H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gallusser A, Kirchhausen T, and Bonifacino JS. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269: 1872-1875, 1995[ISI][Medline].

23.   Oleinikov, AV, Zhao J, and Makker SP. Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin. Biochem J 347: 613-621, 2000[ISI][Medline].

24.   Orlando, RA, and Farquhar MG. Functional domains of the receptor-associated protein (RAP). Proc Natl Acad Sci USA 91: 3161-3165, 1994[Abstract].

25.   Orlando, RA, Rader K, Authier F, Yamazaki H, Posner BI, Bergeron JJ, and Farquhar MG. Megalin is an endocytic receptor for insulin. J Am Soc Nephrol 9: 1759-1766, 1998[Abstract].

26.   Patrie, KM, Drescher AJ, Goyal M, Wiggins RC, and Margolis B. The membrane-associated guanylate kinase protein magi-1 binds megalin and is present in glomerular podocytes. J Am Soc Nephrol 12: 667-677, 2001[Abstract/Free Full Text].

27.   Rader, K, Orlando RA, Lou X, and Farquhar MG. Characterization of ANKRA, a novel ankyrin repeat protein that interacts with the cytoplasmic domain of megalin. J Am Soc Nephrol 11: 2167-2178, 2000[Abstract/Free Full Text].

28.   Rapoport, I, Chen YC, Cupers P, Shoelson SE, and Kirchhausen T. Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif- binding site. EMBO J 17: 2148-2155, 1998[Abstract/Free Full Text].

29.   Rodriguez-Boulan, E, and Gonzalez A. Glycans in post-Golgi apical targeting: sorting signals or structural props? Trends Cell Biol 9: 291-294, 1999[ISI][Medline].

30.   Saito, A, Pietromonaco S, Loo AK, and Farquhar MG. Complete cloning and sequencing of rat gp330/"megalin"; a distinctive member of the low density lipoprotein receptor gene family. Proc Natl Acad Sci USA 91: 9725-9729, 1994[Abstract/Free Full Text].

31.   Saito, A, Yamazaki H, Rader K, Nakatani A, Ullrich R, Kerjaschki D, Orlando RA, and Farquhar MG. Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis. Proc Natl Acad Sci USA 93: 8601-8605, 1996[Abstract/Free Full Text].

32.   Sargiacomo, M, Lisanti M, Graeve L, Le Bivic A, and Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J Membr Biol 107: 277-286, 1989[ISI][Medline].

33.   Scheiffele, P, Peranen J, and Simons K. N-glycans as apical sorting signals in epithelial cells. Nature 378: 96-98, 1995[ISI][Medline].

34.   Short, DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, and Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273: 19797-19801, 1998[Abstract/Free Full Text].

35.   Simons, K, and Ikonen E. Functional rafts in cell membranes. Nature 387: 569-572, 1997[ISI][Medline].

36.   Takeda, T, Go WY, Orlando RA, and Farquhar MG. Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells. Mol Biol Cell 11: 3219-3232, 2000[Abstract/Free Full Text].

37.   Trommsdorff, M, Borg JP, Margolis B, and Herz J. Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein. J Biol Chem 273: 33556-33560, 1998[Abstract/Free Full Text].

38.   Yamazaki, H, Ullrich R, Exner M, Saito A, Orlando RA, Kerjaschki D, and Farquhar MG. All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann nephritis. J Am Soc Nephrol 9: 1638-1644, 1998[Abstract].

39.   Yeaman, C, Grindstaff KK, and Nelson WJ. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev 79: 73-98, 1999[Abstract/Free Full Text].

40.   Zheng, G, Bachinsky DR, Stamenkovic I, Strickland DK, Brown D, Andres G, and McCluskey RT. Organ distribution in rats of two members of the low density lipoprotein receptor gene family, gp330 and LRP/alpa 2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 42: 531-542, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 284(5):C1105-C1113
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society