Departments of 1 Cellular and Molecular Medicine and 2 Pathology, University of California, San Diego, La Jolla, California 92093
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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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.
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RESULTS |
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institutes of Health Grant DK-17724.
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FOOTNOTES |
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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.
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