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• W2021086582 abstract "Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M2 subtype is sorted apically, whereas M3 is targeted basolaterally. Expression of M2/M3 receptor chimeras revealed that a 21-residue sequence, Ser271–Ser291, from the M3 third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M3sorting signal into the homologous regions of M2 was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M3 sorting signal also conferred basolateral targeting to M2 when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M3 basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor α-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M3basolateral sorting signal is dominant over apical signals in M2 and acts in a position-independent manner. The M3 sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors. Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M2 subtype is sorted apically, whereas M3 is targeted basolaterally. Expression of M2/M3 receptor chimeras revealed that a 21-residue sequence, Ser271–Ser291, from the M3 third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M3sorting signal into the homologous regions of M2 was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M3 sorting signal also conferred basolateral targeting to M2 when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M3 basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor α-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M3basolateral sorting signal is dominant over apical signals in M2 and acts in a position-independent manner. The M3 sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors. Madin-Darby canine kidney muscarinic acetylcholine receptor third intracellular loop polymerase chain reaction nucleotides interleukin-2 receptor α-chain phosphate-buffered saline cAMP response element transmembrane domain Targeting of newly synthesized proteins to their correct subcellular locales is essential for cell function. Protein sorting is particularly important in polarized cells such as neurons and epithelia, where cell-surface proteins must be specifically routed to distinct plasma membrane subdomains. The mechanisms responsible for the correct targeting of membrane proteins in polarized cells remain a fundamental question in cell biology. Madin-Darby canine kidney (MDCK)1 epithelial cells provide a widely used and well characterized model system for studies of protein targeting (1Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (391) Google Scholar). Polarized MDCK cells establish apical and basolateral plasma membrane domains with distinct protein and lipid compositions. Many cell-surface proteins contain sorting signals that direct them to the apical or basolateral domain. Apical sorting signals can consist of a glycosylphosphatidylinositol anchor (2Lisanti M.P. Caras I.W. Davitz M.A. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar),N-glycans (3Scheiffele P. Peränen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar, 4Hobert M.E. Kil S.J. Medof M.E. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), or protein sequences in the extracellular, transmembrane, and/or cytoplasmic domains (5Kundu A. Avalos R.T. Sanderson C.M. Nayak D.P. J. Virol. 1996; 70: 6508-6515Crossref PubMed Google Scholar, 6Chuang J.-Z. Sung C.-H. J. Cell Biol. 1998; 142: 1245-1256Crossref PubMed Scopus (135) Google Scholar, 7Muth T.R. Ahn J. Caplan M.J. J. Biol. Chem. 1998; 273: 25616-25627Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 8Tugizov S. Maidji E. Xiao J. Zheng Z. Pereira L. J. Virol. 1998; 72: 7374-7386Crossref PubMed Google Scholar, 9Jacob 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). In contrast, basolateral sorting signals are almost always found in the cytoplasmic domain of transmembrane proteins and frequently contain a critical tyrosine residue, a dihydrophobic motif, a cluster of acidic residues, or a combination of these elements (10Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 11Matter K. Yamamoto E.M. Mellman I. J. Cell Biol. 1994; 126: 991-1004Crossref PubMed Scopus (215) Google Scholar, 12Odorizzi G. Trowbridge I.S. J. Biol. Chem. 1997; 272: 11757-11762Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 13Simmen T. Nobile M. Bonifacino J.S. Hunziker W. Mol. Cell. Biol. 1999; 19: 3136-3144Crossref PubMed Scopus (67) Google Scholar). Although much has been learned about the sorting of single-pass transmembrane proteins, little is known about signals that mediate the targeting of proteins with multiple membrane-spanning domains. Muscarinic acetylcholine receptors (mAChRs) are a family of seven-transmembrane domain, G protein-coupled receptors composed of five distinct subtypes (M1–M5). The M1, M3, and M5 receptors preferentially couple to activation of phospholipase C via the Gq/11 family of G proteins, whereas the M2 and M4 receptors preferentially couple to inhibition of adenylyl cyclase via the Gi/o family (14Wess J. Crit. Rev. Neurobiol. 1996; 10: 69-99Crossref PubMed Scopus (423) Google Scholar). In addition to their biochemical specificities, mAChR subtypes have unique cellular and subcellular distributions (15Caulfield M.P. Pharmacol. Ther. 1993; 58: 319-379Crossref PubMed Scopus (1156) Google Scholar). Muscarinic receptors are asymmetrically distributed in polarized cells such as pancreatic and lacrimal acinar cells (16Tan Y.P. Marty A. Trautmann A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11229-11233Crossref PubMed Scopus (42) Google Scholar, 17Toescu E.C. Lawrie A.M. Petersen O.H. Gallacher D.V. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar), lingual epithelial cells (18Simon S.A. Baggett H.C. Arch. Oral Biol. 1992; 37: 685-690Crossref PubMed Scopus (10) Google Scholar), Xenopus oocytes (19Matus-Leibovitch N. Lupu-Meiri M. Oron Y. Pfluegers Arch. 1990; 417: 194-199Crossref PubMed Scopus (15) Google Scholar, 20Davidson A. Mengod G. Matus-Leibovitch N. Oron Y. FEBS Lett. 1991; 284: 252-256Crossref PubMed Scopus (26) Google Scholar), and MDCK epithelial cells (21Nadler L.S. Kumar G. Hinds T.R. Migeon J.C. Nathanson N.M. Am. J. Physiol. 1999; 277: C1220-C1228Crossref PubMed Google Scholar). Furthermore, mAChR subtypes are differentially localized in a variety of neuronal cells. For example, the M1 receptor is expressed in the cell bodies and dendrites of hippocampal pyramidal neurons and granule cells in the dentate gyrus, where it mediates postsynaptic responses to acetylcholine (22Levey A.I. Edmunds S.M. Koliatsos V. Wiley R.G. Heilman C.J. J. Neurosci. 1995; 15: 4077-4092Crossref PubMed Google Scholar). In contrast, M2 is found mainly in the axon terminals of cholinergic and non-cholinergic septohippocampal projection neurons and hippocampal interneurons, where it modulates neurotransmitter release (23Rouse S.T. Thomas T.M. Levey A.I. Life Sci. 1997; 60: 1031-1038Crossref PubMed Scopus (67) Google Scholar, 24Hájos N. Papp E.C.S. Acsády L. Levey A.I. Freund T.F. Neuroscience. 1998; 82: 355-376Crossref PubMed Scopus (189) Google Scholar). The M3 receptor is found both on cell bodies and dendrites of hippocampal granule and pyramidal neurons and on axon terminals in the hippocampal molecular layer and striatum (25Levey A.I. Edmunds S.M. Heilman C.J. Desmond T.J. Frey K.A. Neuroscience. 1994; 63: 207-221Crossref PubMed Scopus (125) Google Scholar, 26Rouse S.T. Gilmor M.L. Levey A.I. Neuroscience. 1998; 86: 221-232Crossref PubMed Scopus (42) Google Scholar). Despite the differential localization of mAChR subtypes in a variety of polarized cells, little is known about the mechanisms by which their precise subcellular distributions are achieved. To begin to elucidate the signals and mechanisms that govern mAChR targeting, we have utilized the MDCK cell system to identify sorting determinants for mAChR subtypes. Although the follicle-stimulating hormone receptor possesses a basolateral sorting signal in its C-terminal cytoplasmic tail (27Beau I. Groyer-Picard M.-T. Le Bivic A. Vannier B. Loosfelt H. Milgrom E. Misrahi M. J. Biol. Chem. 1998; 273: 18610-18616Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and basolateral targeting information for the α2A-adrenergic receptor appears to be in a domain composed of multiple transmembrane sequences (28Saunders C. Keefer J.R. Bonner C.A. Limbird L.E. J. Biol. Chem. 1998; 273: 24196-24206Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), sorting information for G protein-coupled receptors in polarized cells remains largely unknown. In this report, we used chimeric receptor constructs in a gain-of-function approach to identify a basolateral sorting signal for the M3 mAChR in MDCK cells. The M3 basolateral sorting signal lies in a 21-amino acid sequence from the N-terminal portion of the third intracellular (i3) loop, is dominant over apical signals in the M2 receptor, and can act in a position-independent manner. This M3 sequence represents a novel basolateral sorting motif for G protein-coupled receptors. A modified FLAG epitope (DYKDDDDA) was added to the extracellular N termini of the M1–M5 mAChR coding sequences immediately after the initiator methionines using PCR to generate pFM1, pFM2, pFM3, pFM4, and pFM5. The mouse M1 (29Shapiro R.A. Scherer N.M. Habecker B.A. Subers E.M. Nathanson N.M. J. Biol. Chem. 1988; 263: 18397-18403Abstract Full Text PDF PubMed Google Scholar), porcine M2 (clone Mc7) (30Peralta 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 (334) Google Scholar), human M3 (31Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (657) Google Scholar), human M4 (32Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1219) Google Scholar), and human M5 (31Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (657) Google Scholar) mAChR cDNAs in the mammalian expression vector pCDPS (31Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (657) Google Scholar) were used as templates. For M1, M2, and M4, the forward primer encoded the FLAG epitope, and the forward and reverse primers contained unique restriction sites to facilitate subcloning into pCDPS. PCR fragments were as follows: M1,KpnI-NheI, nt 1–676 of M1 coding sequence; M2, KpnI-MscI, nt 1–689 of M2 coding sequence; and M4,SacI-NheI, nt 1–1229 of M4 coding sequence. The M3 and M5 receptors were FLAG-tagged using a sequential PCR approach as described (33Goldman 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), with the FLAG epitope encoded by internal primers. The M3 PCR product (NcoI-SnaBI) contained 306 nt of pCDPS vector sequence and nt 1–462 of M3 coding sequence. The M5 PCR product (NcoI-EcoRI) contained 367 base pairs of pCDPS vector sequence and nt 1–1021 of M5 coding sequence. PCR products were subcloned into the parental plasmids to generate epitope-tagged mAChRs. The ability of the FLAG-tagged receptors to bind the muscarinic antagonist [3H]quinuclidinyl benzilate (47 Ci/mmol; Amersham Pharmacia Biotech) was verified by transient expression in COS-7 or JEG-3 cells. The presence of the FLAG epitope was then verified by immunoprecipitation from transfected cell membranes using the anti-FLAG M2 monoclonal antibody (Sigma). Studies of FLAG-M2mAChR-mediated inhibition of adenylyl cyclase, receptor desensitization, and sequestration have been reported previously (34Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). M2/M3 chimeric mAChRs were constructed using sequential PCR as described (33Goldman 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 M2coding sequence with the homologous regions of M3 coding sequence as aligned in Ref. 31Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (657) Google Scholar. pFM2, pFM3, or M2/M3 chimeric constructs were used as PCR templates for subsequent chimeras. All PCR-amplified constructs were engineered with BglII and EcoRI sites at their 5′- and 3′-ends, respectively, and cloned into the BglII andEcoRI sites of pCDPS. The sequences comprising the M2/M3 chimeras are as follows, with the numbers in parentheses representing the amino acid residues of M3that were substituted into M2: M2/M3-(240–590), coding nt 1–579 of M2 and nt 694–1779 of M3; M2/M3-(486–590), coding nt 1–1143 of M2 and nt 1456–1779 of M3; M2/M3-(240–485), coding nt 1–579 of M2, nt 694–1455 of M3, and nt 1144–1404 of M2; M2/M3-(384–485), coding nt 1–1014 of M2, nt 1150–1455 of M3, and nt 1144–1404 of M2; M2/M3-(240–383), coding nt 1–579 of M2, nt 694–1149 of M3, and nt 1015–1404 of M2; M2/M3-(240–309), coding nt 1–579 of M2, nt 694–927 of M3, and nt 793–1404 of M2; M2/M3-(253–296), coding nt 1–621 of M2, nt 757–888 of M3, and nt 754–1404 of M2; M2/M3-(297–309), coding nt 1–753 of M2, nt 889–927 of M3, and nt 793–1404 of M2; M2/M3-(253–269), coding nt 1–621 of M2, nt 757–807 of M3, and nt 673–1404 of M2; and M2/M3-(266–296), coding nt 1–660 of M2, nt 796–888 of M3, and nt 754–1404 of M2. Fusion proteins in which M3 sequences were added to either the i3 loop or the C terminus of M2 were generated by sequential PCR using pFM2 and either M2/M3-(266–296) or pFM3 as templates, respectively. The M2+M3-(i3:266–296) PCR product was cloned into the BglII and EcoRI sites of pCDPS, whereas the M2+M3 C-terminal fusion constructs were subcloned into the MscI and EcoRI sites of the parental pFM2 plasmid. The sequences comprising the M2+M3 fusion proteins are as follows: M2+M3-(i3:266–296), coding nt 1–660 of M2, nt 796–888 of M3, and nt 661–1404 of M2; M2+M3-(C-term:266–296), coding nt 1–1398 of M2 and nt 796–888 of M3; M2+M3-(C-term:271–296), coding nt 1–1398 of M2 and nt 811–888 of M3; M2+M3-(C-term:266–291), coding nt 1–1398 of M2 and nt 796–873 of M3; M2+M3-(C-term:271–291), coding nt 1–1398 of M2 and nt 811–873 of M3; and M2+M3-(C-term:570–590), coding nt 1–1398 of M2 and nt 1708–1779 of M3. The interleukin-2 receptor/M3 fusion protein was generated by sequential PCR using the human interleukin-2 receptor α-chain (IL-2Rα; Tac antigen) cDNA (pIL2R3; kindly provided by Dr. Warren J. Leonard, National Institutes of Health, Bethesda, MD) (35Leonard W.J. Depper J.M. Crabtree G.R. Rudikoff S. Pumphrey J. Robb R.J. Krönke M. Svetlik P.B. Peffer N.J. Waldmann T.A. Greene W.C. Nature. 1984; 311: 626-631Crossref PubMed Scopus (605) Google Scholar) and pFM3 as templates. This fusion protein consists of coding nt 796–888 (Ala266–Gln296) of M3fused to the C terminus of IL-2Rα. Both the fusion protein and wild-type IL-2Rα were cloned into the BglII andEcoRI sites of pCDPS. PCR-amplified DNA sequences were verified using an Applied Biosystems Model 373A automated sequencing system. MDCK (strain II) cells were obtained from Dr. Keith Mostov (University of California, San Francisco, CA). JEG-3 human choriocarcinoma and COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). All cell lines were maintained in Dulbecco's modified Eagle's medium 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% CO2environment. To analyze the targeting of mAChR constructs, MDCK cells seeded at near-confluency (3.5 × 105cells/well) on 2-well glass chamber slides (4.2 cm2/well; Nalge Nunc International, Naperville, IL) were transfected the following day using the calcium phosphate precipitation method (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with 4 μg of receptor cDNA/well. Cells were fixed at confluence (36–48 h post-transfection) with paraformaldehyde solution (4% (w/v) paraformaldehyde and 4% (w/v) sucrose in phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, and 1.5 mmKH2PO4), pH 7.4) for 30 min at room temperature and processed for immunocytochemistry. 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 (1.2 μg/ml), anti-IL-2Rα (1:100; Upstate Biotechnology, Inc., Lake Placid, NY), or anti-β-catenin (1:100; Transduction Laboratories, Lexington, KY) monoclonal antibody in PBST containing 3% bovine serum albumin and 0.25% Triton X-100 overnight at 4 °C in a humid chamber. Following four washes with PBST, cells were incubated with fluorescein isothiocyanate-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 four more washes with PBST, slides were coverslipped with Vectashield (Vector Labs, Inc., Burlingame, CA). Staining was visualized using a Nikon Optiphot 2 microscope equipped with a 60× Nikon oil immersion objective. Fluorescent images were collected in both thex-y and x-z planes using a Bio-Rad MRC600 laser scanning confocal microscope. For eachx-y image, a z-series of ∼20 optical sections was taken at 0.7-μm intervals from the apical to the basolateral regions of the cells. Images were projected and analyzed using Adobe Photoshop. Quantitation of the apical/basolateral distributions of mAChR constructs was performed using the public domain NIH Image program (developed at the National Institutes of Health). The mean pixel intensity/unit area (pixel values 0–255) of staining in the apical and basolateral domains was determined by manually outlining the areas of interest in the raw (unprocessed) x-z images. Data were processed using Microsoft Excel. Muscarinic receptor-mediated changes in forskolin-stimulated cAMP levels in transiently transfected JEG-3 cells were analyzed as described previously (37Migeon J.C. Nathanson N.M. J. Biol. Chem. 1994; 269: 9767-9773Abstract Full Text PDF PubMed Google Scholar). Transfection mixtures contained (per well) 30 ng of receptor cDNA, 25 ng of α168-CRE-luciferase plasmid (38Mellon P.L. Clegg C.H. Correll L.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4887-4891Crossref PubMed Scopus (201) Google Scholar), 40 ng of Rous sarcoma virus-β-galactosidase plasmid (39Edlund T. Walker M.D. Barr P.J. Rutter W.J. Science. 1985; 230: 912-916Crossref PubMed Scopus (395) Google Scholar), 100 ng of Gαi2 (40Jones D.T. Reed R.R. J. Biol. Chem. 1987; 262: 14241-14249Abstract Full Text PDF PubMed Google Scholar) in pCDPS, and 55 ng of pCDPS carrier to achieve a total of 250 ng of DNA/well. The medium was changed 20–24 h after transfection; cells were treated with 0.4 μm forskolin and various concentrations of carbamylcholine (carbachol) an additional 20–24 h later as described (41Schlador M.L. Grubbs R.D. Nathanson N.M. J. Biol. Chem. 2000; 275: 23295-23302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and lysed; and assays of luciferase and β-galactosidase activities were performed (37Migeon J.C. Nathanson N.M. J. Biol. Chem. 1994; 269: 9767-9773Abstract Full Text PDF PubMed Google Scholar). Muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis was determined in COS-7 cells as previously described (41Schlador M.L. Grubbs R.D. Nathanson N.M. J. Biol. Chem. 2000; 275: 23295-23302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) using 5 μg of receptor DNA/100-mm dish for transfection. Cell-surface expression of mAChR constructs in transfected JEG-3 cells was determined by the binding ofN-[3H]methylscopolamine, a membrane-impermeable muscarinic antagonist, to intact cells as previously described (41Schlador M.L. Grubbs R.D. Nathanson N.M. J. Biol. Chem. 2000; 275: 23295-23302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) with the following modifications. Transfection mixtures contained (per 100-mm culture dish) 1.2 μg of receptor cDNA, 1.0 μg of α168 CRE-luciferase plasmid, 1.6 μg of Rous sarcoma virus-β-galactosidase plasmid, 4.0 μg of Gαi2, and 2.2 μg of pCDPS carrier to achieve a total of 10.0 μg of DNA/dish. Cells from each dish were subcultured onto one 6-well plate 20–24 h after transfection and allowed to attach for an additional 24 h. N-[3H]Methylscopolamine binding assays were performed as described (41Schlador M.L. Grubbs R.D. Nathanson N.M. J. Biol. Chem. 2000; 275: 23295-23302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), except that protein content was determined by the method of Lowry et al.(42Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Previous studies demonstrated that mAChRs are asymmetrically distributed in a variety of polarized cells (16Tan Y.P. Marty A. Trautmann A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11229-11233Crossref PubMed Scopus (42) Google Scholar, 17Toescu E.C. Lawrie A.M. Petersen O.H. Gallacher D.V. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar, 18Simon S.A. Baggett H.C. Arch. Oral Biol. 1992; 37: 685-690Crossref PubMed Scopus (10) Google Scholar, 19Matus-Leibovitch N. Lupu-Meiri M. Oron Y. Pfluegers Arch. 1990; 417: 194-199Crossref PubMed Scopus (15) Google Scholar, 20Davidson A. Mengod G. Matus-Leibovitch N. Oron Y. FEBS Lett. 1991; 284: 252-256Crossref PubMed Scopus (26) Google Scholar, 21Nadler L.S. Kumar G. Hinds T.R. Migeon J.C. Nathanson N.M. Am. J. Physiol. 1999; 277: C1220-C1228Crossref PubMed Google Scholar, 22Levey A.I. Edmunds S.M. Koliatsos V. Wiley R.G. Heilman C.J. J. Neurosci. 1995; 15: 4077-4092Crossref PubMed Google Scholar). Despite many observations of mAChR localization, little is known concerning the cellular mechanisms and molecular signals that underlie the sorting of mAChRs to specific subcellular domains. To identify sorting signals for mAChR subtypes, we examined their targeting in MDCK epithelial cells, a widely used and well characterized model system for protein sorting. For these studies, recombinant mAChRs were FLAG-tagged at their N termini to enable immunochemical detection. The FLAG-tagged M1, M2, and M3 receptors were expressed at levels similar to their non-tagged counterparts when transfected into COS-7 or JEG-3 cells, whereas the expression of FLAG-M4 and FLAG-M5 was significantly lower than that of the non-tagged receptors. 2L. S. Nadler and N. M. Nathanson, unpublished observations. For receptor targeting studies, the steady-state distributions of recombinant mAChRs were analyzed in confluent MDCK cells by immunocytochemistry and confocal microscopy. The M2 and M3 receptors displayed reciprocal polarized distributions. Although M2 was highly enriched on the apical membrane, M3 was localized to the basolateral domain (Fig.1, B and C). Although some basal M3 staining was evident, most M3 immunoreactivity was restricted to the lateral subdomain. In contrast, the M1, M4, and M5 receptors exhibited non-polarized distributions, with labeling apparent throughout the cells (Fig. 1, A,D, and E). MDCK cell polarity was verified by examining the distribution of the endogenous E-cadherin-associated protein β-catenin, a basolateral marker (4Hobert M.E. Kil S.J. Medof M.E. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 43Yeaman C. Grindstaff K.K. Nelson W.J. Physiol. Rev. 1999; 79: 73-98Crossref PubMed Scopus (444) Google Scholar). β-Catenin was exclusively localized to the lateral subdomain (Fig. 1 F), indicating that the cells are correctly polarized under our experimental conditions. These results demonstrate that the M2 and M3 mAChRs are targeted to opposite domains of MDCK cells at steady state and suggest that they possess sorting signals that direct them to distinct subcellular locations. The differential targeting of the M2 and M3 mAChRs allowed us to test the feasibility of using receptor chimeras to identify regions of the receptors important for either apical sorting of M2 or basolateral targeting of M3. Since basolateral sorting signals can be dominant over apical signals when present in the same molecule (4Hobert M.E. Kil S.J. Medof M.E. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 44Le Bivic A. Sambuy Y. Patzak A. Patil N. Chao M. Rodriguez-Boulan E. J. Cell Biol. 1991; 115: 607-618Crossref PubMed Scopus (116) Google Scholar), we analyzed M2/M3receptor chimeras in a gain-of-function approach to identify regions of M3 sequence that would confer basolateral targeting to the otherwise apical M2 receptor. Schematic diagrams of the initial set of chimeric constructs are presented in Fig.2. Fig. 3shows the steady-state localizations of these hybrid receptors. The first construct, M2/M3-(240–590), contains M3 Phe240–Leu590, encompassing the C-terminal half of the fifth transmembrane domain (TM5), the i3 loop, the sixth and seventh transmembrane domains (TM6 and TM7, respectively), and the C-terminal tail in the context of the M2 receptor. The M2/M3-(240–590) chimera displayed a primarily basolateral localization in MDCK cells similar to wild-type M3 (Fig. 3, B andC). This result suggests that a region of sequence in the C-terminal half of M3 is sufficient for basolateral targeting.Figure 3Localization of M2/M3chimeric receptors in MDCK cells. MDCK cells transfected with constructs encoding FLAG-tagged M2, M3, or M2/M3 receptor chimeras were fixed and stained with anti-FLAG antibody as described under “Experimental Procedures.” x-y (upper panels; projected z-series) and x-z(lower panels; vertical section taken at the level of thewhite line) images for each construct are shown.A, M2; B, M3;C, M2/M3-(240–590); D, M2/M3-(486–590); E, M2/M3-(240–485); F, M2/M3-(384–485); G, M2/M3-(240–383); H, M2/M3-(240–309). Bar = 15 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next sought to identify the sequence containing the basolateral sorting signal by substitution of smaller regions of M3into the homologous positions of M2. M3Glu486–Leu590 did not confer basolateral targeting to M2, with the chimera having an apical distribution very similar to wild-type M2 (Fig. 3,A and D). This indicates that the M3basolateral sorting signal does not lie in TM6 or TM7 or in the C-terminal tail. M2/M3-(240–485), which contains the C-terminal half of TM5 and the i3 loop of M3in the context of M2, did exhibit a primarily basolateral distribution that was very similar to wild-type M3 (Fig. 3,B and E), suggesting that the basolateral sorting signal lies within the M3 i3 loop. Further dissection of the M3 sequence confirmed this possibility. Substitution of M3 Phe240–Leu383, containing the N-terminal half of the i3 loop, into M2 conferred a mainly basolateral localization to the receptor molecule, although minor apical staining was also apparent (Fig. 3 G). In contrast, substitution of M3 Pro384–Lys485, comprising the C-terminal half of the i3 loop, did not confer basolateral localization, and this chimera displayed an apical distribution similar to wild-type M2 (Fig. 3, Aand F). This suggests that the M3 basolateral sorting signal lies in the N-terminal half of the i3 loop. Furthermore, when M3 Phe240–Gly309 was substituted into M2, the receptor exhibited a basolateral distribution virtually indistinguishable from wild-type M3(Fig. 3, B and H). These data indicate that a 70-amino acid region from TM5 and the i3 loop of M3contains a basolateral sorting signal that is s" @default.
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• W2021086582 title "Identification of a Basolateral Sorting Signal for the M3 Muscarinic Acetylcholine Receptor in Madin-Darby Canine Kidney Cells" @default.
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