HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 46, 35381-35396, November 17, 2006

This Article Abstract Full Text (PDF) All Versions of this Article: 281/46/35381    most recent M605954200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chmelar, R. S. Articles by Nathanson, N. M. Search for Related Content PubMed PubMed Citation Articles by Chmelar, R. S. Articles by Nathanson, N. M. Social Bookmarking                      What's this?

Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M₂ Muscarinic Acetylcholine Receptor^*

From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750

Received for publication, June 21, 2006 , and in revised form, September 11, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies have shown that the M₂ receptor is localized at steady state to the apical domain in Madin-Darby canine kidney (MDCK) epithelial cells. In this study, we identify the molecular determinants governing the localization and the route of apical delivery of the M₂ receptor. First, by confocal analysis of a transiently transfected glycosylation mutant in which the three putative glycosylation sites were mutated, we determined that N-glycans are not necessary for the apical targeting of the M₂ receptor. Next, using a chimeric receptor strategy, we found that two independent sequences within the M₂ third intracellular loop can confer apical targeting to the basolaterally targeted M₄ receptor, Val²⁷⁰-Lys²⁸⁰ and Lys²⁸⁰-Ser³⁵⁰. Experiments using Triton X-100 extraction followed by OptiPrep^TM density gradient centrifugation and cholera toxin -subunit-induced patching demonstrate that apical targeting is not because of association with lipid rafts. ³⁵S-Metabolic labeling experiments with domain-specific surface biotinylation as well as immunocytochemical analysis of the time course of surface appearance of newly transfected confluent MDCK cells expressing FLAG-M₂-GFP demonstrate that the M₂ receptor achieves its apical localization after first appearing on the basolateral domain. Domain-specific application of tannic acid of newly transfected cells indicates that initial basolateral plasma membrane expression is required for subsequent apical localization. This is the first demonstration that a G-protein-coupled receptor achieves its apical localization in MDCK cells via transcytosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The biogenesis of membrane polarity is crucial for the development of multicellular organisms and for the proper functioning of polarized cell types such as neurons and epithelial cells. Biochemically and functionally distinct plasma membrane domains are required for cellular processes such as neuronal transmission, migration, solute uptake and transport, secretion, body water homeostasis, and signal transduction. The mechanism by which cells generate and maintain cellular polarity remains a fundamental question of cell biology.

The renal epithelial Madin-Darby canine kidney (MDCK)² cell line provides a model system for the study of protein targeting because of the ability of this cell line, when grown to confluency, to form a polarized monolayer (1). Polarized epithelial cell surface membranes are divided into apical and basolateral domains possessing distinct protein and lipid compositions that are separated by tight junctions. This spatial asymmetry is generated and maintained by one of three pathways. Proteins can be targeted directly to the apical or basolateral membrane from the trans-Golgi network (TGN) via the exocytotic pathway (2). They can also be targeted indirectly by being delivered to one domain, typically the basolateral domain, endocytosed, and then either recycled back to the original membrane or redirected to the opposite domain in a process termed transcytosis (3, 4). Alternatively, proteins can be randomly targeted to both domains and achieve their asymmetric distribution by selective stabilization or retention at one plasma membrane (5-7). Although other epithelial cell types, including hepatocytes and intestinal cells, primarily utilize the indirect pathway for apical protein delivery (8), MDCK cells primarily use the direct pathway (3).

In MDCK cells, newly synthesized apical and basolateral membrane proteins are segregated into separate transport vesicles within the TGN by virtue of sorting signals within the protein (2, 9, 10). The routing of basolaterally targeted proteins expressed in this cell line is mediated by discrete cytosolic amino acid sequences that frequently resemble endocytosis signals. These sequences often contain a critical tyrosine residue within an NPXYorYXX motif (where is a bulky hydrophobic amino acid), a dihydrophobic motif, a cluster of acidic residues, or a combination of these elements (11-13). Recent evidence suggests that in some cases these motifs are recognized by the clathrin adapter proteins AP-1, AP-2, or AP-3 that sequester them into basolateral transport vesicles (3, 14-16). Molecular signals mediating apical transport in MDCK cells are much more diverse. The apical targeting of proteins within MDCK cells can be mediated by the lipid anchor of glycosylphosphatidylinositol (GPI)-anchored proteins, by protein N- or O-linked glycans, or by amino acid residues located in the extracellular, transmembrane, or cytoplasmic domains of proteins (3, 17-19). The prevalent model of apical membrane sorting proposes that apical proteins are sequestered into apical transport vesicles via association with sorting platforms rich in cholesterol and sphingolipid-rich lipid rafts at the level of the trans-Golgi network (20, 21).

The distribution of the mAChR subtypes when exogenously expressed in MDCK cells has been characterized. Although the M₁, M₄, and M₅ receptors appeared to be nontargeted, the M₂ and the M₃ receptors displayed an apical and a basolateral distribution, respectively (22). The M₃ basolateral sorting sequence has been defined in a previous study as a 7-amino acid motif residing in the N-terminal portion of the third intracellular loop (22, 23). In this study, the molecular signals mediating the apical targeting of the M₂ mAChR and the mechanism of delivery to the apical membrane were investigated.

In this study we use M₂/M₄ chimeric receptor constructs to show that there is apical targeting information within the third intracellular loop of the M₂ mAChR. We use sequences from the M₂ third intracellular loop appended to the C terminus of M₄ to define two adjacent sequences of amino acids that were able to confer apical targeting to the M₄ receptor in a position-independent fashion as follows: an 11-amino acid sequence, Val²⁷⁰-Lys²⁸⁰ and a 71-amino acid sequence, Lys²⁸⁰-Ser³⁵⁰. Furthermore, we demonstrate by using deletion analysis that either of these apical sorting sequences is sufficient to direct the apical sorting of the M₂ receptor. To address the mechanism by which the M₂ receptor cargo is sequestered into apically targeted vesicles, we determine whether or not the M₂ receptor is raft-associated by using both biochemical and confocal analyses. Finally, we use metabolic labeling followed by surface biotinylation, immunocytochemical imaging of the cell-surface staining pattern of newly transfected confluent MDCK cells expressing the M₂-GFP, and domain-specific application of the cell-impermeable cross-linking agent tannic acid to demonstrate that the M₂ receptor is initially transported to the basolateral domain prior to its subsequent apical localization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—MDCK (strain II) cells were obtained from Dr. Keith Mostov (University of California, San Francisco). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin sulfate at 37 °C in a humidified 10% CO₂ environment.

Transfection of mAChRs and Chimeric Constructs—To analyze the targeting of mAChR constructs, MDCK cells were seeded at high density (1 x 10⁶ cells/well) on 24.5-mm polycarbonate Transwell filters (0.4-µm pore size; Costar, Cambridge, MA) in DMEM supplemented with 10% fetal bovine serum (1 ml in the apical and 2.5 ml in the basolateral compartments) 24 h before transfection. For each construct to be transfected, 4 µg of DNA was added to a final volume of 100 µl of DMEM (without serum or antibiotics), and 8 µl of Lipofectamine 2000 (1:2 ratio of DNA:Lipofectamine 2000 reagent; Invitrogen) was added to 92 µl of DMEM (without serum or antibiotics). The two solutions were combined and incubated on a bench top for at least 30 min before being added to the medium in the apical compartment of the plated MDCK cells. Twenty four hours after the start of transfection, the wells of transfected cells were replaced with fresh media.

Immunocytochemical Analysis—36-48 h after transfection, cells were rinsed three times with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.5 mM KH₂PO₄, pH 7.4) and fixed with paraformaldehyde solution (4% (w/v) paraformaldehyde and 4% (w/v) sucrose) in PBS for 30 min at room temperature and processed for immunocytochemistry. For permeabilized conditions, fixed cells were rinsed twice with PBST (PBS containing 0.1% (v/v) Tween 20), permeabilized with 0.25% (v/v) Triton X-100 (in PBS) for 5 min at room temperature, and blocked with 10% (w/v) bovine serum albumin in PBST containing 0.25% Triton X-100 for 2 h at room temperature. After blocking, cells were incubated with anti-FLAG M2 monoclonal antibody (2 µg/ml; Sigma) or affinity-purified subtype-specific polyclonal anti-M₂ (1:200) raised to the third intracellular loop of mouse M₂ mAChR (24), in PBST containing 3% bovine serum albumin and 0.25% Triton X-100 overnight at 4 °C in a humid chamber or2hat room temperature. Following three washes with PBST, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (1:250; Cappel Research Products, Durham, NC) in PBST containing 3% bovine serum albumin and 0.25% Triton X-100 for 2-3 h at room temperature. After three more washes with PBST, slides were coverslipped with Vectashield (Vector Laboratories, Inc., Burlingame, CA). For nonpermeabilized conditions, Tween and Triton X-100 were omitted from all solutions listed above.

Fluorescent images were collected in both the x-y and x-z planes using a Leica TCS SP1/NT confocal microscope (Leica Microsystems, Inc., Exton, PA.) using x100, 1.4 N.A. oil immersion lens in the W. M. Keck Imaging Center at the University of Washington. For each x-y image, a projected z-series image was generated by taking images at 0.6-µm intervals from the apical to the basolateral regions of the cells and compressing to a single x-y image. Images were processed using Adobe Photoshop. Quantitation of the apical/basolateral distribution of mAChR deletion constructs was performed using the public domain NIH Image program (developed at the National Institutes of Health). The mean pixel intensity/unit area was determined by manually outlining the areas of interest in the raw (unprocessed) x-z images. Data are expressed as percent basolateral of total staining and are presented as means ± S.E. for the number of experiments indicated. Between 2 and 16 images were quantitated for each experiment. Data were processed using Microsoft Excel.

Construction of Epitope-tagged Chimeric and C-terminal Fusion mAChRs—A modified FLAG epitope (DYKDDDDA) was added to the extracellular N termini of the porcine M₂ (clone Mc7 (25)) and human M₄ (26) mAChR coding sequences immediately after the initiator methionines using PCR as described (22). The M₂ and M₄ receptors were cloned into the KpnI/EcoRI and EcoRV sites of site of pcDNA3.1 (Invitrogen), respectively.

M₂/M₄ chimeric mAChRs were constructed by using sequential PCR using Pfu or Pfu Turbo polymerase (Stratagene) as described previously (27) to replace parts of the M₂ coding sequence with the homologous regions of M₄ coding sequence as described previously. pFM₂, pFM₄, or M₂/M₄ chimeric constructs were used as PCR templates for subsequent chimeras. All PCR-amplified constructs were designed with KpnI and EcoRI sites at their 5'- and 3'-ends, respectively, and ligated into pcDNA3.1. The sequences comprising the M₂/M₄ chimeras are as follows, with the numbers in parentheses representing the amino acid residues of M₂ that were substituted into M₄: M₄/M₂(208-467), coding nucleotides (nt) 1-648 of M₄ and nt 622-1401 of M₂;M₄/M₂(391-467), coding nt 1-1209 of M₄ and nt 1171-1401 of M₂;M₂(1-207)/M₄, coding nt 1-621 of M₂ and nt 649-1440 of M₄;M₂(1-390)/M₄, coding nt 1-1170 of M₂ and nt 1210-1440 of M₄; and M₄/M₂(208-390), coding nt 1-648 and nt 1210-1440 of M₄ and nt 622-1170 of M₂.

C-terminal fusion proteins M₄+M₂(208-252), M₄+M₂(208-280), and M₄+M₂(208-352), in which M₂ sequences were fused to the C terminus of M₄, were generated by sequential PCR using pFM₂ and pFM₄ as a template. The above C-terminal fusion constructs were inserted into the BglII/EcoRI site of pCDPS. The sequences comprising the M₄ C-terminal M₂ appendages are as follows: M₂(208-327), coding nt 622-981; M₂(208-280), coding nt 622-840 of M₂; and M₂(208-252), coding nt 622-756.

C-terminal fusion proteins M₄+M₂(220-280), M₄+M₂(280-350), M₄+M₂(220-260), M₄+M₂(250-270), and M₄+M₂(260-280) were constructed by first generating an M₄ construct (nt 1-1437) that possessed three C-terminal alanine residues (gcg gcc gcc) in place of the amber stop codon to create a NotI restriction site. This construct was inserted into the EcoRV/NotI sites of pcDNA3.1 to create M₄-C'NotI. The following M₂ sequences were PCR-amplified with three N-terminal alanines (gcg gcc gcc) to create a NotI site and a C-terminal XhoI site. The following M₂ sequences were then ligated into the M₄-C'NotI construct in pcDNA3.1 using the NotI/XhoI sites: M₂(220-280), coding nt 658-840; M₂(280-350), coding nt 838-1050; M₂(220-260), coding nt 658-780; M₂(250-270), coding nt 748-810, and M₂(260-280) and coding nt 778-840. The M₄+M₂(270-280), coding nt 808-840, was constructed by including the M₂ nucleotides to be added to the C terminus of M₄ within the 3'-primer, placing the STOP codon of M₄ and terminated with a STOP codon followed by repeated XbaI sites. The 5'-primer was designed to anneal to the M₄ sequence 6 bp upstream through 9 bp downstream (nt 982-1002) of the unique SacII of M₄. The construct was digested with SacII and XbaI and ligated into a similarly digested M₄ construct within PCDNA3.1(+).

A C-terminal M₂-GFP fusion protein was constructed by PCR amplifying FLAG-M₂ with an N-terminal EcoRI site and a C-terminal XbaI site (which replaced the C-terminal ochre stop codon) and ligating the PCR product into pCS2⁺XLT vector (28) that was engineered to produce an in-frame N-terminal GFP fusion protein, pCS2⁺XLT-GFP (obtained from R. T. Moon).

Generation of FLAG-M₂ mAChR Deletion and Glycosylation-defective Mutants—The following M₂ mAChR deletion mutants were generated by sequential PCR as described previously (27) using porcine FLAG-M₂ as a template: M₂(del 220-380), lacking nt 658-1140; M₂(del 227-315), lacking nt 678-945; M₂(del 220-280), lacking nt 658-840; M₂(del 280-325), lacking nt 838-975; and M₂(del 303-380) lacking nt 907-1140. Mutants M₂(del 227-315), M₂del (280-325), and M₂(del 303-380) were generously provided by Dr. Michael Schlador. M₂(del 220-380) and M₂(del 220-280) were cloned into the EcoRI/XbaI pcDNA3.1, whereas the M₂(del 227-315), M₂(del 280-325), and M₂(del 303-380) constructs were cloned into pCDPS. The M₂ mAChR glycosylation mutant was described previously (29).

Generation of MDCKII Cells Stably Expressing FLAG-tagged M₂ mAChR—MDCKII cells stably expressing FLAG-tagged M₂ mAChR were generated by transfecting MDCK II cells cultured in a 10-cm plate at 60-80% confluency using the calcium phosphate precipitation method (30) with 10 µg of the pFM₂ receptor in pcDNA3.1. Beginning at 48 h after transfection, stable transformants were selected by treatment with 1 mg/ml G418 (Geneticin; Invitrogen) for at least 4 weeks following transfection. Drug-resistant cells were selected and then screened by both sterile fluorescence-assisted cell sorting (FACS) and immunocytochemistry. For FACS analysis, cells were dissociated from plates using trypsin (as performed in cell culturing), pelleted by centrifugation, suspended in PBS with 3% BSA and 2 µg/ml monoclonal anti-FLAG antibody, and incubated on ice for 30 min. Cells were again pelleted by centrifugation, rinsed 2x in PBS, and resuspended in PBS with 3% BSA with 1:250 anti-mouse FITC-conjugated secondary antibody, and incubated for 30 min. Cells were rinsed 2x in PBS, resuspended in PBS with 3% BSA, and subjected to FACS analysis (Coulter Elite; Beckman Coulter). Selected cells (75,000) were plated, grown, and split into a 96-well culture dish (Costar) at a dilution of 1 cell/ml. Clones were expanded and further evaluated for FLAG-M₂ receptor expression by immunocytochemistry as described under "Immunocytochemical Analysis."

Detergent Extraction and Flotation in OptiPrep^TM Density Gradients—MDCK cells stably expressing FLAG-tagged M₂ mAChR were grown 3-5 days post-confluency on three 10-cm culture dishes with daily media changes before use. Cells were washed twice in ice-cold PBS and lysed for 30 min in 3 ml of ice-cold TNE (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA), 5 mM dithiothreitol, with 0.5% Triton X-100 and protease inhibitors (Complete; Roche Applied Science) on ice. The lysate was scraped from the dishes, and the plates were rinsed with 1 ml of the ice-cold TNE; the lysates from the three plates were combined and centrifuged at 10,000 x g for 5 min. Membranes were resuspended in 3 ml of the same buffer and were homogenized by passing the sample through a 25-gauge needle 20 times, on ice. The extract was brought to 40% OptiPrep^TM in a final volume of 4 ml and sequentially overlaid with 6 ml of 30% and 2 ml of 5% OptiPrep^TM. Gradients were centrifuged for 18 h at 39,000 rpm at 4 °C in a Beckman SW41Ti rotor. The opalescent band at the 5-30% OptiPrep^TM interface, containing lipid rafts, was collected as the Triton X-100-insoluble fraction, and the 40% OptiPrep^TM layer was harvested as the Triton X-100-soluble fraction. 25-µl aliquots were taken from each 1-ml fraction; 60-80 µl of Laemmli sample buffer was added to each fraction, and the sample was heated for 4 min at 95 °C and subjected to SDS-PAGE on a 10% polyacrylamide gel. Antibodies used were subtype-specific monoclonal anti-M₂ (1:500) as described (31), polyclonal anti-caveolin (1:5000; Transduction Laboratories), and polyclonal anti-human transferrin antibodies (1:200; Santa Cruz Biotechnology, H-300). Renaissance Western blot chemiluminescent reagent (PerkinElmer Life Sciences) was used to detect immunoreactivity.

Quantitation of the distribution of protein in fractions throughout the gradient was performed using the public domain NIH Image program (developed at the National Institutes of Health). The mean pixel intensity/unit area was determined by manually outlining the areas of interest. The percent of total was determined by dividing the pixel intensity of each band by the sum of the intensities of all the bands throughout the gradient. Data were processed using Microsoft Excel; graphing was performed on CA-Cricket Graph III.1.

Cholera Toxin and Antibody-induced Patching—MDCK cells stably expressing FLAG-tagged M₂ mAChR were plated on MDCK cells seeded at 3.5 x 10⁵ cells/well on 2-well glass chamber slides (4.2 cm²/well; Nalge Nunc International) and grown to confluency for 3-4 days. AlexaFluor 594 conjugate of cholera toxin -subunit (ChTx; Molecular Probes) was used to label endogenous glycosphingolipids. Cells were incubated with ChTx (10 µg/ml) in DMEM-HEPES (25 mM HEPES), 0.2% BSA on ice for 30 min and then rinsed three times with ice-cold PBS. For the nonpatched (control) condition, cells were rinsed, fixed, and stained as described under "Immunocytochemical Analysis." Lipid raft aggregation, or patching of ChTx, was induced by incubating the cells with anti-ChTx antibody (1:250 in DMEM-HEPES, 0.2% BSA; Calbiochem-Novabiochem) for 20 min at 37 °C. Cells were removed from the incubator, rinsed, fixed, and stained as described previously. Co-patching of M₂ mAChR and ChTx was performed by simultaneously incubating cells on ice with ChTx and anti-FLAG antibody (2.5 µg/ml) in DMEM-HEPES, 0.2% BSA. After rinsing three times with ice-cold PBS, cells were patched with both anti-ChTx antibody (as described above) and FITC-conjugated goat anti-mouse secondary antibody (1:250; Cappel Research Products, Durham, NC) for 20 min at 37 °C. Cells were rinsed three times with PBS, fixed, and visualized as described above. As a positive control, cells were labeled simultaneously with BODIPY FL-labeled C5 ganglioside GM1 (500 nM on PBS; Molecular Probes) and ChTx for 30 min on ice and were either subsequently fixed or patched as described above with anti-ChTx.

Quantitation of colocalization was performed using the plug-in RG2B_colocalization to ImageJ (version http://rsb.info.nih.gov). The colocalization measurements were taken using a minimum threshold pixel intensity adjusted between 100 and 200 and set equivalently for both channels to correspond to the image intensity. The minimum ratio for pixel intensity between the two channels was set to 0.5. Colocalization pixels were displayed as an image with maximum pixel intensity (255). The RGB output of the plug-in was split into green, red, and blue images, and each image was quantitated by measuring the total pixel area above the threshold intensity used in colocalization determination (see above). Results are displayed as percent colocalization as determined by dividing the area of colocalization pixels by the total pixel area over the threshold of whichever channel was limiting.

Functional Confirmation of Intact Monolayers Prior to Metabolic Labeling—For polarity experiments, MDCKII cells were seeded at a density of 1 x 10⁶ cells/24.5-mm polycarbonate membrane filter (Transwell chambers, 0.4-µm pore size; Costar, Cambridge, MA) and cultured for 5-8 days with medium changes every day. Prior to each functional or immunocytochemical experiment, the integrity of the monolayer was assessed by adding [³H]methoxyinulin (PerkinElmer Life Sciences) to the apical medium and monitoring the leak of [³H]methoxyinulin from the apical compartment to the basolateral compartment by sampling and counting the basolateral medium in a scintillation counter after a 1-h incubation at 37 °C. Chambers with greater than 3% leak per h were discarded.

Metabolic Labeling/Biotinylation Strategy for Determining Surface Delivery of the M₂ mAChR—MDCK cells were metabolically labeled, surface-biotinylated, and evaluated as described (32). MDCK cells stably expressing the FLAG-M₂ receptor cells grown on Transwells were washed twice with PBS and grown for 45 min in methionine/cysteine-free medium (Invitrogen) supplemented with 4 mM glutamine and 5% fetal calf serum. Following removal of this medium, cells were inverted (cell side down), and 150-200 µl of methionine/cysteine-free medium with 5% fetal bovine serum containing 1 µCi/µl ³⁵S-Express protein labeling mixture (NEG-072; PerkinElmer Life Sciences) was added to the bottom of inverted filters. The cells were incubated at 37 °C for various lengths of time. Each time point reflects a pooled sample from three 24.5-mm wells. At the end of the labeling period, cells were washed three times with PBS/CM (PBS containing 0.5 mM CaCl₂ and 2mM MgCl₂). Sulfo-NHS-LC-biotin (Pierce) at 1 mg/ml in PBS buffer was then applied to the apical or basolateral surface and incubated for 30 min on ice in order to covalently label surface proteins. The opposite compartment not being treated with biotin was incubated with PBS/CM containing 1% (w/v) BSA. After the incubation, cells were washed once in 50 mM Tris to quench and three times for 5 min with PBS/CM. Cells were harvested in 1 ml of ice-cold PBS using a rubber policeman and pelleted by centrifugation. Membranes were solubilized by resuspending the pellet in solubilization buffer as follows: 1% digitonin (Calbiochem) and 0.1% cholate (Sigma) in phosphate buffer (10 mM KH₂PO₄, pH 7.0, 1 mM EDTA, 50 mM NaCl) with protease inhibitors (Complete; Roche Applied Science) and rotating at 4 °C for 6 h. Membranes were pelleted by centrifugation, and the supernatant was rotated overnight at 4 °C with 9.0 µg/ml anti-FLAG M₂ monoclonal antibody (Sigma). 50 µl of a 1:1 slurry of protein G-agarose (Roche Applied Science) was added to the membrane/antibody mixture after preincubating the agarose beads for 1 h in 2.5% BSA in solubilization buffer followed by two 5-min washes with solubilization buffer alone and rotated overnight at 4 °C. The next day the protein G-agarose was pelleted by centrifugation. Pellets were washed three times for 5 min with 0.5% digitonin and 0.1% cholate in phosphate buffer with protease inhibitors (Complete; Roche Applied Science). Following the last wash, proteins were eluted from the protein G-agarose beads by incubating in 100 µl of SDS sample buffer at 90 °C for 20 min and agitated every 5 min. A second elution was performed in 50 µl of sample buffer. Samples were combined and subjected to SDS-PAGE on a 10% polyacrylamide gel and subsequent autoradiography using BioMax MS-1 film and a BioMax TransScreen-LE (PerkinElmer Life Sciences). The identity of the radioactive band as that of the M₂ receptor was confirmed by analyzing membranes of M₂ stably expressing MDCK cells by Western analysis using an affinity-purified subtype-specific polyclonal anti-M₂ (1:200) raised to the third intracellular loop of mouse M₂ mAChR and matching the electrophoretic mobility of the immunoreactive band with that of the radioactive band.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Localization of wild type M2 mAChR and a glycosylation-defective M2 receptor mutant transiently transfected into MDCK cells. MDCK cells were transfected with FLAG-tagged M2 mAChR (A) and a site-specific mutant M2 receptor (B) in which the three N-terminal N-glycosylation consensus sites (asparagines 2, 3, and 6) were substituted with the codon for aspartate residues. Cells were fixed, stained, and visualized by confocal microscopy as described under "Experimental Procedures." The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Each image is representative of at least three independent experiments. Bar = 20 µm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Localization of Wild Type M₂ mAChR and a Glycosylation-defective Mutant M₂ Receptor in MDCK Cells—Many studies have indicated that N-or O-glycosylation is critical not only for the exit of newly synthesized proteins from the endoplasmic reticulum (33, 34) and the Golgi (35) but also for targeting of newly synthesized proteins to the correct membrane domain of polarized epithelial cells (35-37). Despite mounting evidence indicating that apical sorting can occur independently of N-glycosylation (18, 38-40), a role for N-glycans in apical sorting continues to be supported by recent evidence that N-glycans alone can mediate apical sorting of the membrane dipeptidase, independent of raft association (41). Furthermore, the addition of glycosylation sites to the basolaterally localized Na,KATPase ₁ resulted in the redirection of this subunit to the apical domain of HGT-1 cells (a human gastric adenocarcinoma cell line) and the redistribution of the associated Na,KATPase ₁ subunit to the apical domain as well (42).

The M₂ mAChR is a sialoglycosylated transmembrane protein (43) that has three potential sites for N-glycosylation. A previous study has shown that N-glycosylation of the M₂ receptor is not required for cell-surface localization or ligand binding and does not confer increased stability against receptor degradation in Chinese hamster ovary cells (29); however, the importance of N-glycans in mediating the membrane targeting of the M₂ receptor within a polarized cell type has not been investigated. To determine whether N-glycosylation plays a role in the steady-state apical localization of the M₂ mAChR in MDCK cells, a glycosylation-defective mutant in which the three asparagine codons were substituted with aspartate residues (29) was transfected into MDCK cells, and the steady-state distribution of the mutant within the polarized monolayer was analyzed by immunocytochemistry and confocal microscopy. Similar to the wild type receptor, the glycosylation-defective mutant was localized to the apical domain of MDCK cells (Fig. 1, A and B, respectively). This result demonstrates that the steady-state distribution of the M₂ mAChR to the apical domain of confluent MDCK cells is independent of N-glycosylation.

The Third Intracellular Loop of the M₂ mAChR Contains Apical Sorting Information—Whereas the M₂ receptor displays an apical distribution when expressed in MDCK cells, the M₄ receptor was shown previously to exhibit an apparently nonpolarized, cytoplasmic distribution under permeabilizing staining conditions when transiently expressed in MDCK cells (22). Because of the differential localization of these receptors and their high degree of amino acid identity (56% identity, 78% if the third intracellular loop is excluded), we tested the use of M₂/M₄ receptor chimeric constructs in a gain-of-function approach to determine which domain of M₂ could confer apical targeting to the M₄ receptor. To visualize the plasma membrane distribution of the M₂ and M₄ receptor constructs, we used nonpermeabilizing conditions to stain for cell-surface expression of the extracellular N-terminal FLAG epitope of transiently transfected filter-grown MDCK cells. Under these conditions, the FLAG-M₄ construct was localized to the basolateral domain on the cell surface (Fig. 3B).

Because of the differential distribution of the M₂ and M₄ receptors on the cell surface, we were able to pursue the approach of defining an M₂ apical sorting signal using M₂/M₄ chimeras. A schematic representation of the chimeric constructs is presented in Fig. 2. Fig. 3 shows the steady-state localizations of the chimeric receptors. The first construct, M₄/M₂ (208-467), contains the N-terminal portion of M₄ through the predicted fifth transmembrane (TM5) region and M₂ sequence from the N terminus of the third intracellular loop to the C terminus of the receptor. This chimera displayed an apical distribution in MDCK cells, similar to that of the wild type M₂ receptor (Fig. 3, A and C). This result suggested that a region C-terminal to TM5 of the M₂ receptor is sufficient to confer apical sorting to the M₄ receptor. To test this hypothesis, we examined the surface distribution of a receptor that contained the M₄ sequence through the M₄ third intracellular loop, and M₂ sequence from TM6 through the C terminus of the receptor, M₄/M₂(391-467). As shown in Fig. 3D, this receptor was basolaterally targeted, suggesting that the M₂ apical targeting information was contained in the third intracellular loop of M₂.To further examine this possibility, a pair of constructs representing the inverse of the constructs tested above was generated. These constructs consisted of the N-terminal M₂ sequence either up to M₂(1-207)/M₄ or through M₂(1-390)/M₄, the M₂ third intracellular loop within the M₄ context. In support of the hypothesis that the M₂ third intracellular loop contains apical sorting information, the M₂(1-207)/M₄, which lacked the M₂ third intracellular loop, displayed a basolateral steady-state distribution, whereas the M₂(1-390)/M₄ construct, which encompassed the M₂ third intracellular loop, was apically directed (Fig. 3, E and F). To determine whether the third intracellular loop alone was sufficient to confer apical targeting to the M₄ receptor, the M₂ third intracellular loop sequence was substituted into the context of the M₄ receptor in the construct M₄/M₂(208-390). The steady-state distribution of M₄/M₂(208-390) was apical (Fig. 3G). These results suggest that the M₂ third intracellular loop contains sufficient sorting information to redirect the basolaterally targeted M₄ receptor to the apical domain in MDCK cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Schematic representation of M2/M4 receptor chimeras. Receptor chimeras were derived from the parental M4 (white) and M2 (black) mAChRs as described under "Experimental Procedures." Regions containing M2 receptor sequences are denoted in black, and regions representing M4 sequences are denoted in white. Numbers in parentheses represent amino acids of the M2 receptor that were substituted into the homologous region of the M4 receptor.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Differential localization of M2/M4 chimeric receptors in MDCK cells. Filter-grown MDCK cells were transiently transfected with FLAG-tagged receptor chimeras, fixed, stained, and visualized by confocal microscopy as described under "Experimental Procedures." The upper panels are images taken in the x-y plane (projected z-series) and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. A, M2 mAChR; B, M4 mAChR; C, M4/M2(208-467); D, M4/M2(391-467); E, M2(1-207)/M4; F, M2(1-390)/M4; and G, M4/M2(208-390). Each image is representative of at least three independent experiments. Bar = 20 µm.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. The M2 apical sorting sequence is position-independent and lies within amino acids 208-280 of the M2 third intracellular loop. A, schematic representation of the M2 third intracellular loop and the localization of constructs containing the sequences (bars) of amino acids (numbers) from the M2 third intracellular loop appended to the C terminus of the M4 mAChR. B, filter-grown MDCK cells were transfected with FLAG-tagged constructs encoding the following: panel 1, M2 receptor; panel 2, M4 receptor; panel 3, M4+C-terminal addition of M2(208-327); panel 4, M4+C-terminal addition of M2(208-280); and panel 5, M4+C-terminal addition of M2(208-252). Cells were fixed, stained, and visualized by confocal microscopy as described under "Experimental Procedures." The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Each image is representative of at least three independent experiments. Bar = 20 µm.

Attempts at further defining the apical sorting sequence within Lys²⁸⁰-Ser³⁵⁰ by dividing it into smaller sequences and appending these to the C terminus of the M₄ receptor resulted in constructs that did not express well.³ It was therefore not possible to determine whether a smaller region of this sequence was sufficient to confer apical targeting. Nonetheless, these results indicate that there are redundant apical targeting sequences within the third intracellular loop of M₂ and that M₂ amino acids 270-280 and 280-350 each contain sufficient sorting information to confer apical targeting to the M₄ receptor. Both apical sequences are indicated in Fig. 5C.

Disruption of Both M₂ Apical Sorting Sequences 270-280 and 280-350 Results in Basolateral Accumulation of the M₂ Receptor—Several constructs containing the M₂ third intracellular loop deletions (diagrammed in Fig. 6A) were evaluated to determine whether sequences localized to this region were also necessary for the apical targeting of the M₂ receptor. First, to determine whether deletion of the M₂ third intracellular loop was sufficient to cause mis-localization of the receptor, amino acids 220-380 (comprising 89% of the third intracellular loop) were deleted. Although less than 10% (8.0 ± 4.4%, n = 6, where n is the number of independent transfections, between 2 and 14 cells were quantitated in each experiment; mean ± S.E.) of the total staining for the wild type receptor was present in the basolateral domain, the deletion mutant receptor M₂(del 220-380) displayed a significant increase in basolateral staining (30.8 ± 10.1%, n = 5). A series of mutants containing smaller overlapping deletions was generated to determine the necessity of those sequences for the apical targeting of the M₂ receptor. Although deletion mutants M₂(del 220-280), M₂(del 280-325), and M₂(del 303-380) displayed little or no increase in the amount of basolateral staining of the receptor when compared with wild type (10.4 ± 4.6%, n = 2; 10.5 ± 4.1%, n = 5; 23.0 ± 5.7, n = 6), deletion of amino acids 227-315, which disrupts both apical sorting sequences, resulted in the largest degree of basolateral accumulation (40.5 ± 6.4%, n = 5). These results support the finding that the M₂ receptor contains redundant sorting information because either sorting sequence was sufficient to direct the receptor apically, and only when both were deleted was there significant mis-localization of the receptor.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. M2 amino acids 270-280 and 280-350 contain autonomous apical sorting signals. A, schematic representation of the M2 third intracellular loop and the localization of constructs containing the sequences (bars) of amino acids (numbers) from the M2 third intracellular loop appended to the C terminus of the M4 mAChR. B, filter-grown MDCK cells were transfected with FLAG-tagged constructs encoding the following: panel 1, M4+C-terminal addition of M2(220-280); panel 2, M4+C-terminal addition of M2(280-350); panel 3, M4+C-terminal addition of M2(220-260); panel 4, M4+C-terminal addition of M2(250-270); panel 5, M4+C-terminal addition of M2(260-280); and panel 6, M4+C-terminal addition of M2(270-280). Cells were fixed, stained, and visualized by confocal microscopy as described under "Experimental Procedures." The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Each image is representative of at least three independent experiments. Bar = 20 µm. C, apical sorting sequences of the M2 mAChR, amino acids 270-280 and 280-350.

The M₂ Receptor Is Not Found in Cholera Toxin-induced Patches—Because resistance to solubilization in the nonionic detergent Triton X-100 as a method for determining raft association has been criticized as an indirect method open to a variety of interpretations that may not truly reflect raft size, structure, composition, or even existence (54, 55), we used cross-linking of ChTx patches to obtain visual evidence of M₂ association with lipid rafts in intact cells. MDCK cells stably expressing the FLAG-M₂ mAChR were labeled with the AlexaFluor-conjugated ChTx-594. The pentavalent cholera toxin-subunit binds glycosphingolipids and exhibits a particularly strong affinity for the GM1 glycosphingolipid, a widely used indicator for the distribution of lipid rafts (56). Cholera toxin binding to five cell surface GM1 molecules leads to co-clustering of various glycosylated raft-associated proteins that are further aggregated, or "patched," by exposure to anti-ChTx antibody, which brings entire lipid microdomains together into patches (21, 57).

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Deletion of M2 amino acids 227-316 compromises the ability of M2 to achieve its apical localization. A, schematic representation of the M2 third intracellular loop and the deletions (indicated by lines) of the amino acids (numbers) of each M2 deletion construct. B, filter-grown MDCK cells were transfected with FLAG-tagged constructs encoding the following: panel 1, M2; panel 2, M2(del 220-380); panel 3, M2(del 220-280); panel 4, M2(del 227-316); panel 5, M2(del 303-380); and panel 6, M2(del 280-325). Cells were fixed, stained, and visualized by confocal microscopy as described under "Experimental Procedures." The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Each image is representative of at least three independent experiments. Bar = 20 µm.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Lack of lipid raft association of the M2 mAChR as determined by Triton X-100 extraction followed by OptiPrepTM gradient centrifugation. A, MDCK cells stably expressing the M2 mAChR were extracted with Triton X-100 on ice, and extracted membranes were subjected to an OptiPrepTM multistep gradient. Fractions were collected from the top and were analyzed by Western blotting as described under "Experimental Procedures." Each image is representative of two other experiments. B, quantitative analyses of the protein present in each fraction of the gradient. The intensity of each band was quantitated using NIH Image and expressed as a percentage of the total.

View larger version (38K): [in this window] [in a new window]   FIGURE 8. Lack of lipid raft association of the M2 mAChR as determined by cholera toxin patching. A, patching of BODIPY GM1 ganglioside. MDCK cells stably expressing FLAG-tagged M2 were simultaneously labeled with ChTx-594 and BODIPY fluorescently labeled ganglioside. Cells were then either fixed (panel 1) or patched (panel 2) with anti-ChTx antibody, fixed, and then visualized by confocal microscopy as described under "Experimental Procedures." The 1st column indicates BODIPY fluorescence; the 2nd column indicates ChTx-594 fluorescence, and the 3rd column is an overlay of the first two columns. B, MDCK cells stably expressing FLAG-tagged M2 were labeled with the AlexaFluor 594-conjugated cholera toxin (ChTx-594) subunit. Cells were then either fixed, stained with mouse anti-FLAG antibody, and labeled with FITC-conjugated anti-mouse antibody without patching (control) (panel 3); patched with anti-ChTx antibody, fixed, stained with mouse anti-FLAG antibody, and labeled with FITC-conjugated anti-mouse antibody (panel 4), or treated with mouse anti-FLAG antibody and co-patched by treatment with both anti-ChTx and FITC-conjugated anti-mouse antibodies (panel 5). Cells were then visualized by confocal microscopy as described under "Experimental Procedures." The 1st column indicates FITC fluorescence, the 2nd column indicates ChTx-594 fluorescence, and the 3rd column is an overlay of the first two columns. Further x5 magnifications of the white squares present in each of the merged images are presented to the right. Each image is representative of at least two other experiments. Bar = 20 µm. C, colocalization of fluorophores for each experimental condition was quantitated as described under "Experimental Procedures" and expressed as percent colocalization.

View larger version (13K): [in this window] [in a new window]   FIGURE 9. Metabolic labeling/biotinylation strategy for determining surface delivery of the M2 mAChR. The amount of newly synthesized receptor delivered to the apical versus basolateral surface was quantified by biotinylating one surface or the other of metabolically labeled MDCKII cells stably expressing the FLAG-M2 mAChR in Transwell culture and subsequently isolating radiolabeled receptor on the biotinylated surface through sequential streptavidin-agarose and immunoprecipitation. Cells were surface-biotinylated on either the apical (Ap) or basolateral (Bl) surface after metabolically labeling with 35S-Express protein labeling mixture for the times indicated as follows: 15, 30, and 60 min. The control lane (C) represents MDCK cells metabolically labeled for 2 h that were not surface-biotinylated and were otherwise treated similarly to other samples. The molecular mass marker at 75 kDa is indicated.

To determine the pathway by which the M₂ mAChR achieves its steady-state apical distribution when expressed in MDCK cells, FLAG-M₂ stably expressing cells were metabolic labeled and surface-biotinylated, and proteins were immunoprecipitated with anti-FLAG antibody, followed by streptavidin precipitation, and analyzed by SDS-PAGE, followed by autoradiography. As shown in Fig. 9, at 15 min, newly synthesized epitope-tagged M₂ mAChR was first detected on the basolateral surface, whereas the amount of receptor present on the apical domain was similar to that of control (representing nonbiotinylated samples). At 30 min, the majority of newly synthesized epitope-tagged M₂ mAChR remained localized at the basolateral surface (83.6 ± 13.7%, n = 2 independent experiments); however, significant apical accumulation was observed. By 60 min the receptor showed a greater accumulation on the apical domain when compared with the basolateral domain (60.0 ± 3.8%, n = 2). The identity of the band as the M₂ receptor was ensured by concurrently subjecting a membrane sample of the same M₂ stably expressing MDCK cells to SDS-PAGE and matching the migration of the immunoreactive M₂ band (using a monoclonal anti-M₂ antibody) with that of the radioactive signal.³ These results suggest that the M₂ mAChR accumulates initially on the basolateral surface of MDCK cells, and within an hour the majority of the receptor is present on the apical domain.

Time Course of Expression of the M₂ mAChR in Newly Transfected MDCK Cells as Monitored by Confocal Microscopy—As discussed above, the metabolic labeling studies indicate that the M₂ mAChR accumulates initially within the basolateral domain of MDCK cells. As a further test of this result, the fusion protein FLAG-tagged M₂-GFP was transiently transfected into MDCK cells. The surface expression of the newly synthesized M₂ receptor was analyzed by staining (red) for the FLAG epitope under nonpermeabilizing conditions, whereas GFP fluorescence allows visualization of total cellular M₂ expression. Because preliminary experiments indicated that the receptor started to appear on the cell surface 12 h after the start of transfection, the time course of receptor expression was determined using 12 h as the initial time point. Although the expression level varied among cells, only the highest expressing cells were analyzed at the stated time points. Twelve hours after transfection (t = 0; Fig. 10), the surface staining of very faint M₂-GFP expressing cells was undetectable above background staining. Within 30 min (t = 30; Fig. 10), the surface distribution of the receptor in the most highly expressing cells appeared basolateral. By 60 min, the apical domain was enriched with M₂ receptor compared with expression within the basolateral domain (t = 60; Fig. 10). At 120 min, the M₂ distribution appeared apical in highly expressing MDCK cells (t = 120; Fig. 10). However, if the gain at which the confocal images were collected was unaltered from that which was used at earlier time points, the image corresponding to t = 120 min did indeed display M₂ immunoreactivity basolaterally as well.³ The gain was lowered for collection at these later time points in order to avoid overexposure. Images were collected at similar gains for both the green and red channels to reflect what was observed using the microscope alone. The lack of yellow in the overlay projection at earlier time points is because of the intensity differences between the two labels. To ensure that surface staining was not artifactual, an analogous experiment was performed by transiently transfecting FLAG-M₂ in MDCK cells and monitoring the time course of appearance of cell surface staining as described above using a fluorescein-conjugated secondary antibody, which yielded similar results.³ These results corroborate the results obtained by metabolic labeling and surface biotinylation discussed above, and again indicate basolateral surface expression of the M₂ receptor prior to appearance in the apical membrane.

View larger version (52K): [in this window] [in a new window]   FIGURE 10. Time course of surface expression of M2-GFP. Filter-grown MDCK cells were transfected with FLAG-tagged M2-GFP, fixed at t = 0 (panel 1) when surface expression was first observed (8 h after transfection), t = 30 min (panel 2), t = 60 (panel 3), and t = 120 min (panel 4). Cells were stained with mouse anti-FLAG antibody, labeled with Alexa-568 conjugated secondary antibody, and visualized using confocal microscopy as described under "Experimental Procedures." The 1st column indicates GFP fluorescence; the 2nd column indicates Alexa-568 fluorescence, and the 3rd column is an overlay of the first two columns. The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Each image is representative of at least two other experiments. Bar = 20 µm.

The physical integrity and polarization of the MDCK monolayer following treatment with tannic acid were confirmed by staining for basolaterally targeted -catenin. The x-z view indicates that in MDCK cells transfected with M₂-GFP (green), treated for 2 h with tannic acid on the apical domain and stained for -catenin (red), the integrity of the monolayer is maintained and the basolateral distribution of -catenin remains unperturbed (Fig. 11C). Similar results were obtained for cells treated with tannic acid on the basolateral domain.³

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The goal of this study was to characterize the molecular signals involved in the apical targeting of the M₂ mAChR in MDCK cells and address the mechanism by which it achieves its steady-state distribution. The apical targeting of a transmembrane protein in MDCK cells can be mediated by a variety of signals, including glycosylation motifs and amino acid sequences located within the extracellular, transmembrane, or intracellular domain (18, 35, 63, 64). Both N- and O-glycans are postulated to mediate apical targeting in a number or proteins (35, 36, 65, 66) via incorporation into lipid rafts via putative raft-associated carbohydrate-binding lectins localized to the TGN (20, 67). However, there are examples where apical targeting may depend on glycosylation, yet be independent of raft association (68, 69). We found that glycosylation of the M₂ receptor is not required for its targeting in MDCK cells.

View larger version (66K): [in this window] [in a new window]   FIGURE 11. Time course of surface expression of M2-GFP after tannic acid treatment of the apical or basolateral domains of filter-grown MDCK cells. Filter-grown MDCK cells were transfected with FLAG-tagged M2-GFP and fixed after tannic acid treatment of either the apical compartment (A) or basolateral compartment (B) for t = 0 min (panel 1) (control, no treatment) when surface expression was first observed (8 h after transfection), or t = 30 min (panel 2), t = 60 min (panel 3), or t = 120 min (panel 4). Cells were stained with mouse anti-FLAG antibody, labeled with Alexa-568-conjugated secondary antibody, and visualized using confocal microscopy as described under "Experimental Procedures." The 1st column indicates GFP fluorescence; the 2nd column indicates Alexa-568 fluorescence, and the 3rd column is an overlay of the first two columns. C, -catenin staining of tannic acid-treated MDCK cells transfected with M2-GFP. Filter grown MDCK cells transfected with FLAG-tagged M2-GFP were treated for 2 h with 0.5% tannic acid on the apical or the basolateral domain. Cells were fixed, labeled with anti--catenin antibody, stained with Alexa-568-conjugated secondary antibody, and visualized using confocal microscopy as described under "Experimental Procedures." Images shown are for cells treated apically; identical images were obtained for cells treated basolaterally. The upper panels are images taken in the x-y plane (projected z-series), and the lower panels are x-z images of a vertical section taken at or near the midpoint of the image. Bar = 20 µm.

The apical targeting determinants of the M₂ receptor were able to override the basolateral sorting information contained in the M₄ receptor. Although a hierarchy of strength in targeting signals has been demonstrated for a number transmembrane proteins in which deletion of a basolateral targeting sequence reveals the existence of a recessive apical targeting determinant (45, 70, 71), there are studies demonstrating that an apical sorting sequence can override a basolateral targeting sequence as demonstrated here and by others (19, 72-74). Interestingly, in a previous study, the M₂ targeting sequence was not able to override the M₃ basolateral targeting determinant when this M₃ determinant was appended to the C terminus of the M₂ receptor (22). These results suggest that there is no specific hierarchy in the strength of basolateral compared with apical sorting sequences in MDCK cells, but rather that the strength of the signal may be an inherent property of the signal itself.

A series of M₂ third intracellular loop deletion mutants suggested that there are multiple regions within the M₂ third intracellular loop that contribute to its apical targeting. Because deletion of nearly the entire third intracellular loop did not result in a fully unpolarized distribution of the receptor, other sequences besides those investigated here may contribute to the apical targeting of the M₂ receptor. The notion that a protein carries multiple autonomous and redundant targeting motifs is not novel. Examples include the transforming growth factor- precursor (47), the low density lipoprotein receptor (11), and the kidney AE1 anion exchanger (75). Consistent with this idea, the M₃ mAChR receptor also displays similar redundancy of targeting signals as deletion of the primary basolateral targeting sequence identified in the M₃ receptor did not perturb its steady-state basolateral distribution (22).

It is particularly interesting that the sorting sequences of the two muscarinic receptors examined to date contain sorting determinants localized to their third intracellular loops. The majority of polytopic membrane proteins, including the G-protein-coupled receptors rhodopsin, the metabotropic glutamate receptor, and the serotonin 5-HT1B receptor, whose expression pattern has been characterized in MDCK cells, is targeted by motifs localized to their C-terminal domains (46, 72, 76-84). A few multipass transmembrane proteins with cytosolic targeting sequences present exceptions to this paradigm. The calcium ATPase type 2 splice form contains a 45-amino acid insert within the first intracellular loop that confers apical targeting to an otherwise basolaterally targeted protein (85), the multidrug resistance protein 1 contains basolateral targeting information in its third cytoplasmic loop (86), and the liver Na⁺-taurocholate cotransporting polypeptide and prominin retain their basolateral and apical distribution, respectively, despite C-terminal truncation (73, 87). To date, none of the previously identified apical targeting sequences demonstrate homology or amino acid similarity to the targeting sequences identified within the M₂ mAChR. Whether structural similarity is conserved between apical sorting determinants has yet to be determined.

The apical sorting of many transmembrane and GPI-anchored proteins in MDCK cells is mediated by their incorporation into rafts (49, 51). Raft incorporation can occur via the association of GPI anchors with the exoplasmic leaflet of the plasma membrane (88), via N-or O-glycans present on the extracellular domains of proteins (35, 37), or via amino acids within the transmembrane domain or membrane-proximal regions of integral membrane proteins (89, 90). The relevance of raft association for apical targeting depends on the protein being investigated. In the case of the p75 neurotrophin receptor, the presence and location of membrane-proximal O-glycosylation is critical for both raft association and apical delivery (91). In some proteins, such as hemagglutinin, neuraminidase, and megalin, sequences mediating apical sorting are distinct from amino acids involved in raft association, and raft association alone is not sufficient to mediate apical targeting of these proteins (89, 90). Still other apically targeted membrane proteins such as CD3 (63), the dipeptidyl peptidase IV (92), the glycine transporter GLYT2 (69), the glutamate transporter excitatory amino acid transporter (93), the metabotropic glutamate receptor 1b (78), and the guanylyl cyclase C (76) are not raft-associated as determined by Triton X-100 extraction. In this study, by both Triton X-100 extraction and antibody cross-linking of cholera toxin-induced patches, the M₂ mAChR was determined to be minimally associated with lipid rafts. This finding suggests that the mechanism by which the M₂ mAChR receptor is sequestered into apically targeted vesicles at the level of the TGN is not via direct association with rafts. For this protein and other nonraft associated polytopic membrane proteins possessing cytosolic apical targeting domains, the mechanism by which the protein is concentrated in apically targeted cargo vesicles has yet to be determined. Given the evidence presented here, it is indeed possible that this subset of proteins is packaged with basolaterally targeted proteins and redirected apically after achieving a basolateral membrane distribution.

Proteins can achieve their final membrane localization through one of three pathways in epithelial cells: direct delivery, selective stabilization after random delivery, and via transcytosis to one plasma membrane domain following delivery to the opposite domain (3). The data presented here demonstrate that by metabolic ³⁵S labeling and surface immunoprecipitation, as well as confocal analysis of newly transfected confluent MDCK cells, that the M₂ mAChR is first directed to the basolateral domain before it accumulates on the apical domain. This represents the first report of a seven-transmembrane receptor undergoing transcytosis in MDCK cells. For some epithelial cell types other than MDCK cells, transcytosis is a predominant mechanism by which proteins achieve their apical localization (94). Although in MDCK cells most newly synthesized apical and basolateral proteins are targeted directly from the TGN to their final surface localization, the Na⁺/K⁺-ATPase (58) and the _2B-adrenergic receptor (6) achieve their final basolateral localization after random surface delivery and selective stabilization at the basolateral domain, and the polymeric immunoglobulin A receptor (59, 60) is targeted directly to the basolateral surface of MDCK cells and subsequently achieves its apical localization via transcytosis. Indeed, there are a number of other proteins utilizing the transcytotic pathway in MDCK cells (40, 95). Whether the initial basolateral localization of these proteins is because of infidelity in apical targeting or is reflective of a physiological role for transcytosis is not known.

The data presented here show that the apical accumulation of the M₂ mAChR occurs subsequent to its basolateral localization. ³⁵S labeling and surface biotinylation assays of stably transfected MDCK cells demonstrate that the M₂ receptor accumulates at the basolateral surface starting 15 min after radioactive label is incorporated into newly synthesized receptor. The M₂ receptor only starts to appreciably accumulate over background levels at the apical surface 30 min after receptors are synthesized, with the majority achieving apical localization 1 h after incorporation of the radiolabel. By immunocytochemical methods, the expression of newly transfected FLAG-tagged M₂-GFP in MDCK cells is also first detected in the basolateral domain. Only at subsequent time points does apical surface expression of the M₂ receptor become detectable.

Polishchuk et al. (62) used domain-specific treatment with tannic acid to selectively block vesicle fusion and protein delivery to either the apical or basolateral domain of MDCK cells. Blocking fusion of transport vesicles to the apical domain resulted in the basolateral accumulation of the M₂ receptor, whereas blocking fusion of transport vesicles to the basolateral domain blocked the appearance of the receptor at both the basolateral and apical domains. These results are consistent with the results of both the metabolic labeling and time course analyses shown in Fig. 9 and provide further support for a requirement for basolateral transport and transcytosis prior to the ultimate transport of the M₂ mAChR to the apical membrane. Blocking basolateral fusion of cargo vesicles by tannic acid resulted in the lack of any surface expression of the M₂ receptor. These results suggest that basolateral surface expression is prerequisite to subsequent apical surface accumulation of the receptor and that there is a post-plasma membrane sorting mechanism for at least some apically targeted proteins in MDCK cells. We also attempted to determine whether the apical targeting determinant located within amino acids 270-280 contains solely apical targeting information or whether this sequence was required for the basolateral transit of the receptor. Unfortunately, receptor expression of the M₂ C-terminal appendages to the M₄ mAChR was not sufficiently strong to observe pre-steady-state accumulation at either membrane domain at time points earlier than 24 h post-transfection.³

The conclusions of the work of Polishchuk et al. (62) have recently been criticized by Paladino et al. (96), who suggested that membrane polarity is not fully achieved at 3 days in culture and that treatment with tannic acid can interfere with segregation of the apical and basolateral domains. However, our experiments reveal that polarization is achieved within the limits of our time course by the apical and basolateral localization of the M₂ receptor and -catenin, respectively, as shown in Fig. 11C. Furthermore, the metabolic labeling and domain-specific biotinylation experiments were performed on stably transfected cells 5 days after initiation of polarization, and provide independent confirmation of the transcytosis of the M₂ receptor.

In conclusion, the results presented here demonstrate the M₂ mAChR is an apically targeted protein that achieves its localization independent of raft association and that basolateral localization is prerequisite to apical accumulation. This is the first reported example of a seven-transmembrane receptor that is predominantly localized to the apical surface by undergoing transcytosis from the basolateral domain in MDCK cells. This may represent a model mechanism utilized by other proteins for their localization in MDCK cells as well as other polarized cell types.

	FOOTNOTES

 
^* This work was supported by Grant R01NS26920 from the National Institutes of Health. 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.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington School of Medicine, Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso{at}u.washington.edu.

² The abbreviations used are: MDCK, Madin-Darby canine kidney; TGN, trans-Golgi network; GPI, glycosylphosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% (v/v) Tween 20; FITC, fluorescein isothiocyanate; nt, nucleotides; FACS, fluorescence assisted cell sorting; ChTx, cholera toxin -subunit; TM, transmembrane; mAChR, muscarinic acetylcholine receptor; BSA, bovine serum albumin; GFP, green fluorescent protein.

³ R. S. Chmelar and N. M. Nathanson, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Paulette Brunner and Greg Martin for training and assistance on the confocal microscope and Michael Schlador for several of the M₂ deletion mutant constructs and intellectual contributions.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Rodriguez-Boulan, E., and Powell, S. K. (1992) Annu. Rev. Cell Biol. 8, 395-427[CrossRef][Medline] [Order article via Infotrieve]
2. Keller, P., Toomre, D., Diaz, E., White, J., and Simons, K. (2001) Nat. Cell Biol. 3, 140-149[CrossRef][Medline] [Order article via Infotrieve]
3. Mostov, K. E., Verges, M., and Altschuler, Y. (2000) Curr. Opin. Cell Biol. 12, 483-490[CrossRef][Medline] [Order article via Infotrieve]
4. Aroeti, B., Okhrimenko, H., Reich, V., and Orzech, E. (1998) Biochim. Biophys. Acta 1376, 57-90[Medline] [Order article via Infotrieve]
5. Gut, A., Balda, M. S., and Matter, K. (1998) J. Biol. Chem. 273, 29381-29388[Abstract/Free Full Text]
6. Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017-5024[Abstract/Free Full Text]
7. Matter, K. (2000) Curr. Biol. 10, R 39-R42[CrossRef][Medline] [Order article via Infotrieve]
8. Bastaki, M., Braiterman, L. T., Johns, D. C., Chen, Y. H., and Hubbard, A. L. (2002) Mol. Biol. Cell 13, 225-237[Abstract/Free Full Text]
9. Kreitzer, G., Schmoranzer, J., Low, S. H., Li, X., Gan, Y., Weimbs, T., Simon, S. M., and Rodriguez-Boulan, E. (2003) Nat. Cell Biol. 5, 126-136[CrossRef][Medline] [Order article via Infotrieve]
10. Simons, K., and Wandinger-Ness, A. (1990) Cell 62, 207-210[CrossRef][Medline] [Order article via Infotrieve]
11. Matter, K., Hunziker, W., and Mellman, I. (1992) Cell 71, 741-753[CrossRef][Medline] [Order article via Infotrieve]
12. Matter, K., Yamamoto, E. M., and Mellman, I. (1994) J. Cell Biol. 126, 991-1004[Abstract/Free Full Text]
13. Odorizzi, G., and Trowbridge, I. S. (1997) J. Biol. Chem. 272, 11757-11762[Abstract/Free Full Text]
14. Orzech, E., Schlessinger, K., Weiss, A., Okamoto, C. T., and Aroeti, B. (1999) J. Biol. Chem. 274, 2201-2215[Abstract/Free Full Text]
15. Folsch, H., Pypaert, M., Schu, P., and Mellman, I. (2001) J. Cell Biol. 152, 595-606[Abstract/Free Full Text]
16. Rapoport, I., Chen, Y. C., Cupers, P., Shoelson, S. E., and Kirchhausen, T. (1998) EMBO J. 17, 2148-2155[CrossRef][Medline] [Order article via Infotrieve]
17. Rodriguez-Boulan, E., and Gonzalez, A. (1999) Trends Cell Biol. 9, 291-294[CrossRef][Medline] [Order article via Infotrieve]
18. Lin, S., Naim, H. Y., Rodriguez, A. C., and Roth, M. G. (1998) J. Cell Biol. 142, 51-57[Abstract/Free Full Text]
19. Jacob, R., Preuss, U., Panzer, P., Alfalah, M., Quack, S., Roth, M. G., Naim, H., and Naim, H. Y. (1999) J. Biol. Chem. 274, 8061-8067[Abstract/Free Full Text]
20. Fullekrug, J., and Simons, K. (2004) Ann. N. Y. Acad. Sci. 1014, 164-169[CrossRef][Medline] [Order article via Infotrieve]
21. Schuck, S., and Simons, K. (2004) J. Cell Sci. 117, 5955-5964[Abstract/Free Full Text]
22. Nadler, L. S., Kumar, G., and Nathanson, N. M. (2001) J. Biol. Chem. 276, 10539-10547[Abstract/Free Full Text]
23. Iverson, H. A., Fox, D., III, Nadler, L. S., Klevit, R. E., and Nathanson, N. M. (2005) J. Biol. Chem. 280, 24568-24575[Abstract/Free Full Text]
24. Hamilton, S. E., Loose, M. D., Qi, M., Levey, A. I., Hille, B., McKnight, G. S., Idzerda, R. L., and Nathanson, N. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13311-13316[Abstract/Free Full Text]
25. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I., and Capon, D. J. (1987) Science 236, 600-605[Abstract/Free Full Text]
26. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. (1987) Science 237, 527-532[Abstract/Free Full Text]
27. Goldman, P. S., Schlador, M. L., Shapiro, R. A., and Nathanson, N. M. (1996) J. Biol. Chem. 271, 4215-4222[Abstract/Free Full Text]
28. Rupp, R. A., Snider, L., and Weintraub, H. (1994) Genes Dev. 8, 1311-1323[Abstract/Free Full Text]
29. van Koppen, C. J., and Nathanson, N. M. (1990) J. Biol. Chem. 265, 20887-20892[Abstract/Free Full Text]
30. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 16.30-16.40, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
31. Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6896[CrossRef][Medline] [Order article via Infotrieve]
32. Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340-11347[Abstract/Free Full Text]
33. Green, R. F., Meiss, H. K., and Rodriguez-Boulan, E. (1981) J. Cell Biol. 89, 230-239[Abstract/Free Full Text]
34. Roth, M. G., Fitzpatrick, J. P., and Compans, R. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6430-6434[Abstract/Free Full Text]
35. Gut, A., Kappeler, F., Hyka, N., Balda, M. S., Hauri, H. P., and Matter, K. (1998) EMBO J. 17, 1919-1929[CrossRef][Medline] [Order article via Infotrieve]
36. Scheiffele, P., Peranen, J., and Simons, K. (1995) Nature 378, 96-98[CrossRef][Medline] [Order article via Infotrieve]
37. Benting, J. H., Rietveld, A. G., and Simons, K. (1999) J. Cell Biol. 146, 313-320[Abstract/Free Full Text]
38. Kundu, A., Avalos, R. T., Sanderson, C. M., and Nayak, D. P. (1996) J. Virol. 70, 6508-6515[Abstract]
39. Marzolo, M. P., Bull, P., and Gonzalez, A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1834-1839[Abstract/Free Full Text]
40. Meerson, N. R., Bello, V., Delaunay, J. L., Slimane, T. A., Delautier, D., Lenoir, C., Trugnan, G., and Maurice, M. (2000) J. Cell Sci. 113, 4193-4202[Abstract]
41. Pang, S., Urquhart, P., and Hooper, N. M. (2004) J. Cell Sci. 117, 5079-5086[Abstract/Free Full Text]
42. Vagin, O., Turdikulova, S., and Sachs, G. (2005) J. Biol. Chem. 280, 43159-43167[Abstract/Free Full Text]
43. Peterson, G. L., Rosenbaum, L. C., Broderick, D. J., and Schimerlik, M. I. (1986) Biochemistry 25, 3189-3202[CrossRef][Medline] [Order article via Infotrieve]
44. Casanova, J. E., Apodaca, G., and Mostov, K. E. (1991) Cell 66, 65-75[CrossRef][Medline] [Order article via Infotrieve]
45. Hunziker, W., Harter, C., Matter, K., and Mellman, I. (1991) Cell 66, 907-920[CrossRef][Medline] [Order article via Infotrieve]
46. Cheng, C., Glover, G., Banker, G., and Amara, S. G. (2002) J. Neurosci. 22, 10643-10652[Abstract/Free Full Text]
47. Dempsey, P. J., Meise, K. S., and Coffey, R. J. (2003) Exp. Cell Res. 285, 159-174[CrossRef][Medline] [Order article via Infotrieve]
48. Saunders, C., Keefer, J. R., Bonner, C. A., and Limbird, L. E. (1998) J. Biol. Chem. 273, 24196-24206[Abstract/Free Full Text]
49. Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve]
50. Ikonen, E., and Simons, K. (1998) Semin. Cell Dev. Biol. 9, 503-509[CrossRef][Medline] [Order article via Infotrieve]
51. Keller, P., and Simons, K. (1998) J. Cell Biol. 140, 1357-1367[Abstract/Free Full Text]
52. Prydz, K., and Simons, K. (2001) Biochem. J. 357, 11-15[CrossRef][Medline] [Order article via Infotrieve]
53. Schmidt, K., Schrader, M., Kern, H. F., and Kleene, R. (2001) J. Biol. Chem. 276, 14315-14323[Abstract/Free Full Text]
54. Munro, S. (2003) Cell 115, 377-388[CrossRef][Medline] [Order article via Infotrieve]
55. Heerklotz, H. (2002) Biophys. J. 83, 2693-2701[Medline] [Order article via Infotrieve]
56. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998) J. Cell Biol. 141, 929-942[Abstract/Free Full Text]
57. Janes, P. W., Ley, S. C., and Magee, A. I. (1999) J. Cell Biol. 147, 447-461[Abstract/Free Full Text]
58. Hammerton, R. W., Krzeminski, K. A., Mays, R. W., Ryan, T. A., Wollner, D. A., and Nelson, W. J. (1991) Science 254, 847-850[Abstract/Free Full Text]
59. Mostov, K. E., and Deitcher, D. L. (1986) Cell 46, 613-621[CrossRef][Medline] [Order article via Infotrieve]
60. Breitfeld, P. P., Casanova, J. E., Harris, J. M., Simister, N. E., and Mostov, K. E. (1989) Methods Cell Biol. 32, 329-337[Medline] [Order article via Infotrieve]
61. Newman, T. M., Robichaud, A., and Rogers, D. F. (1996) Am. J. Respir. Cell Mol. Biol. 15, 529-539[Abstract]
62. Polishchuk, R., Di Pentima, A., and Lippincott-Schwartz, J. (2004) Nat. Cell Biol. 6, 297-307[CrossRef][Medline] [Order article via Infotrieve]
63. Alonso, M. A., Fan, L., and Alarcon, B. (1997) J. Biol. Chem. 272, 30748-30752[Abstract/Free Full Text]
64. Dunbar, L. A., Aronson, P., and Caplan, M. J. (2000) J. Cell Biol. 148, 769-778[Abstract/Free Full Text]
65. Yeaman, C., Le Gall, A. H., Baldwin, A. N., Monlauzeur, L., Le Bivic, A., and Rodriguez-Boulan, E. (1997) J. Cell Biol. 139, 929-940[Abstract/Free Full Text]
66. Spodsberg, N., Alfalah, M., and Naim, H. Y. (2001) J. Biol. Chem. 276, 46597-46604[Abstract/Free Full Text]
67. Rajendran, L., and Simons, K. (2005) J. Cell Sci. 118, 1099-1102[Free Full Text]
68. Zheng, X., Lu, D., and Sadler, J. E. (1999) J. Biol. Chem. 274, 1596-1605[Abstract/Free Full Text]
69. Martinez-Maza, R., Poyatos, I., Lopez-Corcuera, B., Nunez, E., Gimenez, C., Zafra, F., and Aragon, C. (2001) J. Biol. Chem. 276, 2168-2173[Abstract/Free Full Text]
70. Mostov, K. E., de Bruyn Kopsqq, A., and Deitcher, D. L. (1986) Cell 47, 359-364[CrossRef][Medline] [Order article via Infotrieve]
71. Hobert, M., and Carlin, C. (1995) J. Cell Physiol. 162, 434-446[CrossRef][Medline] [Order article via Infotrieve]
72. Inukai, K., Shewan, A. M., Pascoe, W. S., Katayama, S., James, D. E., and Oka, Y. (2004) Mol. Endocrinol. 18, 339-349[Abstract/Free Full Text]
73. Sun, A. Q., Ananthanarayanan, M., Soroka, C. J., Thevananther, S., Shneider, B. L., and Suchy, F. J. (1998) Am. J. Physiol. 275, G 1045-G1055
74. Monlauzeur, L., Rajasekaran, A., Chao, M., Rodriguez-Boulan, E., and Le Bivic, A. (1995) J. Biol. Chem. 270, 12219-12225[Abstract/Free Full Text]
75. Adair-Kirk, T. L., Dorsey, F. C., and Cox, J. V. (2003) J. Cell Sci. 116, 655-663[Abstract/Free Full Text]
76. Hodson, C. A., Ambrogi, I. G., Scott, R. O., Mohler, P. J., and Milgram, S. L. (2006) Traffic 7, 456-464[CrossRef][Medline] [Order article via Infotrieve]
77. Chuang, J. Z., and Sung, C. H. (1998) J. Cell Biol. 142, 1245-1256[Abstract/Free Full Text]
78. Francesconi, A., and Duvoisin, R. M. (2002) J. Neurosci. 22, 2196-2205[Abstract/Free Full Text]
79. Jolimay, N., Franck, L., Langlois, X., Hamon, M., and Darmon, M. (2000) J. Neurosci. 20, 9111-9118[Abstract/Free Full Text]
80. Karim-Jimenez, Z., Hernando, N., Biber, J., and Murer, H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2916-2921[Abstract/Free Full Text]
81. Muth, T. R., Ahn, J., and Caplan, M. J. (1998) J. Biol. Chem. 273, 25616-25627[Abstract/Free Full Text]
82. Brown, A., Muth, T., and Caplan, M. (2004) Am. J. Physiol. 286, C 1071-C1077
83. Regeer, R. R., and Markovich, D. (2004) Am. J. Physiol. 287, C 365-C372
84. Nies, A. T., Konig, J., Cui, Y., Brom, M., Spring, H., and Keppler, D. (2002) Eur. J. Biochem. 269, 1866-1876[Medline] [Order article via Infotrieve]
85. Chicka, M. C., and Strehler, E. E. (2003) J. Biol. Chem. 278, 18464-18470[Abstract/Free Full Text]
86. Westlake, C. J., Qian, Y. M., Gao, M., Vasa, M., Cole, S. P., and Deeley, R. G. (2003) Biochemistry 42, 14099-14113[CrossRef][Medline] [Order article via Infotrieve]
87. Corbeil, D., Roper, K., Hannah, M. J., Hellwig, A., and Huttner, W. B. (1999) J. Cell Sci. 112, 1023-1033[Abstract]
88. Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544[CrossRef][Medline] [Order article via Infotrieve]
89. Barman, S., Ali, A., Hui, E. K., Adhikary, L., and Nayak, D. P. (2001) Virus Res. 77, 61-69[CrossRef][Medline] [Order article via Infotrieve]
90. Marzolo, M. P., Yuseff, M. I., Retamal, C., Donoso, M., Ezquer, F., Farfan, P., Li, Y., and Bu, G. (2003) Traffic 4, 273-288[Medline] [Order article via Infotrieve]
91. Breuza, L., Garcia, M., Delgrossi, M. H., and Le Bivic, A. (2002) Exp. Cell Res. 273, 178-186[CrossRef][Medline] [Order article via Infotrieve]
92. Slimane, T. A., Lenoir, C., Bello, V., Delaunay, J. L., Goding, J. W., Chwetzoff, S., Maurice, M., Fransen, J. A., and Trugnan, G. (2001) Exp. Cell Res. 270, 45-55[CrossRef][Medline] [Order article via Infotrieve]
93. Butchbach, M. E., Tian, G., Guo, H., and Lin, C. L. (2004) J. Biol. Chem. 279, 34388-34396[Abstract/Free Full Text]
94. Bartles, J. R., and Hubbard, A. L. (1988) Trends Biochem. Sci. 13, 181-184[CrossRef][Medline] [Order article via Infotrieve]
95. Burgos, P. V., Klattenhoff, C., de la Fuente, E., Rigotti, A., and Gonzalez, A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 3845-3850[Abstract/Free Full Text]
96. Paladino, S., Pocard, T., Catino, M. A., and Zurzolo, C. (2006) J. Cell Biol. 172, 1023-1034[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				O. Vagin, J. A. Kraut, and G. Sachs Role of N-glycosylation in trafficking of apical membrane proteins in epithelia Am J Physiol Renal Physiol, March 1, 2009; 296(3): F459 - F469. [Abstract] [Full Text] [PDF]

				W. Pradidarcheep, W. T. Labruyere, N. F. Dabhoiwala, and W. H. Lamers Lack of Specificity of Commercially Available Antisera: Better Specifications Needed J. Histochem. Cytochem., December 1, 2008; 56(12): 1099 - 1111. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 281/46/35381    most recent M605954200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chmelar, R. S. Articles by Nathanson, N. M. Search for Related Content PubMed PubMed Citation Articles by Chmelar, R. S. Articles by Nathanson, N. M. Social Bookmarking                      What's this?