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• W1979344319 abstract "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™ density gradient centrifugation and cholera toxin β-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. 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™ density gradient centrifugation and cholera toxin β-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. 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) 2The 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; ChTxβ, cholera toxin β-subunit; TM, transmembrane; mAChR, muscarinic acetylcholine receptor; BSA, bovine serum albumin; GFP, green fluorescent protein.2The 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; ChTxβ, cholera toxin β-subunit; TM, transmembrane; mAChR, muscarinic acetylcholine receptor; BSA, bovine serum albumin; GFP, green fluorescent protein. 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 (1Rodriguez-Boulan E. Powell S.K. Annu. Rev. Cell Biol. 1992; 8: 395-427Crossref PubMed Scopus (353) Google Scholar). 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 (2Keller P. Toomre D. Diaz E. White J. Simons K. Nat. Cell Biol. 2001; 3: 140-149Crossref PubMed Scopus (369) Google Scholar). 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 (3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (331) Google Scholar, 4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar). Alternatively, proteins can be randomly targeted to both domains and achieve their asymmetric distribution by selective stabilization or retention at one plasma membrane (5Gut A. Balda M.S. Matter K. J. Biol. Chem. 1998; 273: 29381-29388Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 6Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Matter K. Curr. Biol. 2000; 10: R39-R42Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Although other epithelial cell types, including hepatocytes and intestinal cells, primarily utilize the indirect pathway for apical protein delivery (8Bastaki M. Braiterman L.T. Johns D.C. Chen Y.H. Hubbard A.L. Mol. Biol. Cell. 2002; 13: 225-237Crossref PubMed Scopus (58) Google Scholar), MDCK cells primarily use the direct pathway (3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (331) Google Scholar).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 (2Keller P. Toomre D. Diaz E. White J. Simons K. Nat. Cell Biol. 2001; 3: 140-149Crossref PubMed Scopus (369) Google Scholar, 9Kreitzer G. Schmoranzer J. Low S.H. Li X. Gan Y. Weimbs T. Simon S.M. Rodriguez-Boulan E. Nat. Cell Biol. 2003; 5: 126-136Crossref PubMed Scopus (184) Google Scholar, 10Simons K. Wandinger-Ness A. Cell. 1990; 62: 207-210Abstract Full Text PDF PubMed Scopus (420) Google Scholar). 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 NPXYorYXXΦ motif (where Φ is a bulky hydrophobic amino acid), a dihydrophobic motif, a cluster of acidic residues, or a combination of these elements (11Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 12Matter K. Yamamoto E.M. Mellman I. J. Cell Biol. 1994; 126: 991-1004Crossref PubMed Scopus (212) Google Scholar, 13Odorizzi G. Trowbridge I.S. J. Biol. Chem. 1997; 272: 11757-11762Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 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 (3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (331) Google Scholar, 14Orzech E. Schlessinger K. Weiss A. Okamoto C.T. Aroeti B. J. Biol. Chem. 1999; 274: 2201-2215Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Folsch H. Pypaert M. Schu P. Mellman I. J. Cell Biol. 2001; 152: 595-606Crossref PubMed Scopus (207) Google Scholar, 16Rapoport I. Chen Y.C. Cupers P. Shoelson S.E. Kirchhausen T. EMBO J. 1998; 17: 2148-2155Crossref PubMed Scopus (257) Google Scholar). 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 (3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (331) Google Scholar, 17Rodriguez-Boulan E. Gonzalez A. Trends Cell Biol. 1999; 9: 291-294Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 18Lin S. Naim H.Y. Rodriguez A.C. Roth M.G. J. Cell Biol. 1998; 142: 51-57Crossref PubMed Scopus (164) Google Scholar, 19Jacob R. Preuss U. Panzer P. Alfalah M. Quack S. Roth M.G. Naim H. Naim H.Y. J. Biol. Chem. 1999; 274: 8061-8067Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 (20Fullekrug J. Simons K. Ann. N. Y. Acad. Sci. 2004; 1014: 164-169Crossref PubMed Scopus (102) Google Scholar, 21Schuck S. Simons K. J. Cell Sci. 2004; 117: 5955-5964Crossref PubMed Scopus (248) Google Scholar).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 (22Nadler L.S. Kumar G. Nathanson N.M. J. Biol. Chem. 2001; 276: 10539-10547Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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 (22Nadler L.S. Kumar G. Nathanson N.M. J. Biol. Chem. 2001; 276: 10539-10547Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 23Iverson H.A. Fox D. Nadler III, L.S. Klevit R.E. Nathanson N.M. J. Biol. Chem. 2005; 280: 24568-24575Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). 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 PROCEDURESCell 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 × 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 (24Hamilton S.E. Loose M.D. Qi M. Levey A.I. Hille B. McKnight G.S. Idzerda R.L. Nathanson N.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13311-13316Crossref PubMed Scopus (308) Google Scholar), 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 ×100, 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 (25Peralta E.G. Winslow J.W. Peterson G.L. Smith D.H. Ashkenazi A. Ramachandran J. Schimerlik M.I. Capon D.J. Science. 1987; 236: 600-605Crossref PubMed Scopus (333) Google Scholar)) and human M4 (26Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1216) Google Scholar) mAChR coding sequences immediately after the initiator methionines using PCR as described (22Nadler L.S. Kumar G. Nathanson N.M. J. Biol. Chem. 2001; 276: 10539-10547Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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 (27Goldman P.S. Schlador M.L. Shapiro R.A. Nathanson N.M. J. Biol. Chem. 1996; 271: 4215-4222Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) 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 (28Rupp R.A. Snider L. Weintraub H. Genes Dev. 1994; 8: 1311-1323Crossref PubMed Scopus (564) Google Scholar) 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 (27Goldman P.S. Schlador M.L. Shapiro R.A. Nathanson N.M. J. Biol. Chem. 1996; 271: 4215-4222Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) 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 (29van Koppen C.J. Nathanson N.M. J. Biol. Chem. 1990; 265: 20887-20892Abstract Full Text PDF PubMed Google Scholar).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 (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.30-16.40Google Scholar) 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 2× 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 2× 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™ 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 × 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™ in a final volume of 4 ml and sequentially overlaid with 6 ml of 30% and 2 ml of 5% OptiPrep™. 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™ interface, containing lipid rafts, was collected as the Triton X-100-insoluble fraction, and the 40% OptiPrep™ 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 (31Luetje C.W. Brumwell C. Norman M.G. Peterson G.L. Schimerlik M.I. Nathanson N.M. Biochemistry. 1987; 26: 6892-6896Crossref PubMed Scopus (29) Google Scholar), 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 × 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 β-subunit (ChTxβ; Molecular Probes) was used to label endogenous glycosphingolipids. Cells were incubated with ChTxβ (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 ChTxβ, was induced by incubating the cells with anti-ChTxβ 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 ChTxβ was performed by simultaneously incubating cells on ice with ChTxβ 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-ChTxβ 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 ChTxβ for 30 min on ice and were either subsequently fixed or patched as described above with anti-ChTxβ.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 × 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 (32Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). MDCK cells stably expressing the FLAG-M2 receptor cells grown on Transwells were washed twice with PBS and grown for 45 min in methio" @default.
• W1979344319 created "2016-06-24" @default.
• W1979344319 creator A5008389963 @default.
• W1979344319 creator A5072567683 @default.
• W1979344319 date "2006-11-01" @default.
• W1979344319 modified "2023-09-30" @default.
• W1979344319 title "Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M2 Muscarinic Acetylcholine Receptor" @default.
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