Identification of a Basolateral Sorting Signal for the M3 Muscarinic Acetylcholine Receptor in Madin-Darby Canine Kidney Cells*

Laurie S. Nadler, Geetha Kumar, and Neil M. NathansonDagger

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

Received for publication, August 8, 2000, and in revised form, November 20, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES

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 M3 sorting 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 alpha -chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M3 basolateral 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES

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 (1). 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 (2), N-glycans (3, 4), or protein sequences in the extracellular, transmembrane, and/or cytoplasmic domains (5-9). 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 (10-13). 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 (14). In addition to their biochemical specificities, mAChR subtypes have unique cellular and subcellular distributions (15). Muscarinic receptors are asymmetrically distributed in polarized cells such as pancreatic and lacrimal acinar cells (16, 17), lingual epithelial cells (18), Xenopus oocytes (19, 20), and MDCK epithelial cells (21). 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 (22). 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 (23, 24). 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 (25, 26). 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 (27), and basolateral targeting information for the alpha 2A-adrenergic receptor appears to be in a domain composed of multiple transmembrane sequences (28), 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES

Construction of Epitope-tagged and Chimeric mAChRs-- 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 (29), porcine M2 (clone Mc7) (30), human M3 (31), human M4 (32), and human M5 (31) mAChR cDNAs in the mammalian expression vector pCDPS (31) 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 (33), 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-M2 mAChR-mediated inhibition of adenylyl cyclase, receptor desensitization, and sequestration have been reported previously (34).

M2/M3 chimeric mAChRs were constructed using sequential PCR as described (33) to replace parts of the M2 coding sequence with the homologous regions of M3 coding sequence as aligned in Ref. 31. 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 and EcoRI sites of pCDPS. The sequences comprising the M2/M3 chimeras are as follows, with the numbers in parentheses representing the amino acid residues of M3 that 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 alpha -chain (IL-2Ralpha ; Tac antigen) cDNA (pIL2R3; kindly provided by Dr. Warren J. Leonard, National Institutes of Health, Bethesda, MD) (35) and pFM3 as templates. This fusion protein consists of coding nt 796-888 (Ala266-Gln296) of M3 fused to the C terminus of IL-2Ralpha . Both the fusion protein and wild-type IL-2Ralpha were cloned into the BglII and EcoRI sites of pCDPS. PCR-amplified DNA sequences were verified using an Applied Biosystems Model 373A automated sequencing system.

Cell Culture-- 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% CO2 environment.

Transfection and Immunocytochemical Analysis of Chimeric mAChR Constructs-- To analyze the targeting of mAChR constructs, MDCK cells seeded at near-confluency (3.5 × 105 cells/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 (36) 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 mM KH2PO4), 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-2Ralpha (1:100; Upstate Biotechnology, Inc., Lake Placid, NY), or anti-beta -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 the x-y and x-z planes using a Bio-Rad MRC600 laser scanning confocal microscope. For each x-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.

Functional Assays-- Muscarinic receptor-mediated changes in forskolin-stimulated cAMP levels in transiently transfected JEG-3 cells were analyzed as described previously (37). Transfection mixtures contained (per well) 30 ng of receptor cDNA, 25 ng of alpha 168-CRE-luciferase plasmid (38), 40 ng of Rous sarcoma virus-beta -galactosidase plasmid (39), 100 ng of Galpha i2 (40) 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 (41) and lysed; and assays of luciferase and beta -galactosidase activities were performed (37). Muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis was determined in COS-7 cells as previously described (41) using 5 µg of receptor DNA/100-mm dish for transfection.

N-[3H]Methylscopolamine Binding Assays-- Cell-surface expression of mAChR constructs in transfected JEG-3 cells was determined by the binding of N-[3H]methylscopolamine, a membrane-impermeable muscarinic antagonist, to intact cells as previously described (41) with the following modifications. Transfection mixtures contained (per 100-mm culture dish) 1.2 µg of receptor cDNA, 1.0 µg of alpha 168 CRE-luciferase plasmid, 1.6 µg of Rous sarcoma virus-beta -galactosidase plasmid, 4.0 µg of Galpha i2, 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 (41), except that protein content was determined by the method of Lowry et al. (42).

    áRESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES

Differential Localization of mAChR Subtypes in MDCK Cells-- Previous studies demonstrated that mAChRs are asymmetrically distributed in a variety of polarized cells (16-22). 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.2

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 beta -catenin, a basolateral marker (4, 43). beta -Catenin was exclusively localized to the lateral subdomain (Fig. 1F), 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.


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Fig. 1.   Differential localization of mAChR subtypes in MDCK cells. MDCK cells were transfected with cDNA encoding the FLAG-tagged M1 (A), M2 (B), M3 (C), M4 (D), or M5 (E) mAChR as described under "Experimental Procedures." Cells were fixed, stained with the anti-FLAG antibody, and visualized by confocal microscopy. Untransfected cells were stained with an antibody against the endogenous basolateral protein beta -catenin (F). For each set of images, the upper panel shows images collected in the x-y plane (projected z-series), and the lower panel shows a vertical (x-z) section taken at the level of the white line in the corresponding x-y image. Bar = 15 µm.

The N-terminal Portion of the M3 Third Intracellular Loop Contains a Basolateral Sorting Signal-- 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 (4, 10, 44), we analyzed M2/M3 receptor 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. 3 shows 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 and C). This result suggests that a region of sequence in the C-terminal half of M3 is sufficient for basolateral targeting.


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Fig. 2.   Schematic representation of M2/M3 receptor chimeras. The M2 (white) and M3 (black) mAChRs were used as the parent constructs from which M2/M3 chimeric receptors were derived as described under "Experimental Procedures." For each chimeric construct, M2 sequence is represented in white and M3 sequence in black. Numbers in parentheses represent the amino acids of M3 that were substituted into the homologous region of the M2 receptor.


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Fig. 3.   Localization of M2/M3 chimeric 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 the white 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.

We next sought to identify the sequence containing the basolateral sorting signal by substitution of smaller regions of M3 into the homologous positions of M2. M3 Glu486-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 M3 basolateral 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 M3 in 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. 3G). 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, A and 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 M3 contains a basolateral sorting signal that is sufficient to redirect the M2 receptor to the basolateral domain of MDCK cells.

Having identified a region of M3 sequence containing a putative basolateral sorting signal, we next wanted to test the role of the transmembrane residues in basolateral targeting. As shown in Fig. 4 (C and D), M2/M3-(253-296), which contains TM5 from M2, displayed a basolateral localization similar to wild-type M3, suggesting that transmembrane residues are not necessary for basolateral targeting. This observation allowed us to focus on residues located in the N-terminal portion of the M3 i3 loop to identify a minimal sequence that provides basolateral targeting information. Since M3 Arg253-Gln296 confers basolateral targeting, we also tested Gln297-Gly309 for basolateral sorting activity. As shown in Fig. 4 (B and E), M2/M3-(297-309) exhibited an apical distribution similar to wild-type M2, suggesting that the basolateral targeting activity lies within M3 Arg253-Gln296. M3 Arg253-Gln269 did not confer basolateral targeting to M2 (Fig. 4F), with the chimera having an apical distribution. However, M2/M3-(266-296) displayed a primarily basolateral localization very similar to wild-type M3 (Fig. 4, C and G). These results strongly suggest that M3 Ala266-Gln296 contains a basolateral sorting signal that is sufficient to redirect the apical M2 receptor.


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Fig. 4.   M3 Ala266-Gln296 contains a basolateral sorting signal. A, presented is a schematic diagram of the N-terminal juxtamembrane region of the M3 i3 loop showing Arg253-Gly309 in single-letter code. Lines and numbers represent sequences and specific residues, respectively, of M3 that were substituted into the homologous regions of M2 to create the chimeras shown in D-G. AP, apical; BL, basolateral. B-G, MDCK cells transfected with constructs encoding FLAG-tagged M2, M3, or M2/M3 chimeras were 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 the white line) images for each construct are shown. B, M2; C, M3; D, M2/M3-(253-296); E, M2/M3-(297-309); F, M2/M3-(253-269); G, M2/M3-(266-296). Bar = 15 µm.

The M3 Sorting Signal Confers Basolateral Targeting to Both M2 and IL-2Ralpha and Is Position-independent-- The results above show that M3 Ala266-Gln296 contains a signal that, when substituted into the M2 receptor, can redirect this apical receptor to the basolateral domain of MDCK cells. However, basolateral targeting of the substitution constructs could be due either to addition of a basolateral signal from M3 or to removal of an apical signal from M2. To distinguish between these possibilities, M3 Ala266-Gln296 was added either to the homologous position in the i3 loop of M2 (M2+M3-(i3:266-296)) or at the C terminus, following the last amino acid of the M2 coding sequence (M2+M3-(C-term:266-296)). When analyzed by confocal microscopy, both of these receptors showed a predominantly basolateral distribution similar to wild-type M3 (Fig. 5, B-D). Thus, the redirection of M2 is due to addition of the M3 basolateral sorting signal rather than elimination of apical targeting information. Furthermore, the data show that the M3 basolateral sorting signal acts in a position-independent manner and is dominant over any apical targeting information present in the M2 receptor.


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Fig. 5.   The M3 basolateral sorting signal is position-independent and can be transferred to a heterologous protein. MDCK cells transfected with constructs encoding FLAG-tagged M2 (A), M3 (B), M2+M3-(i3:266-296) (C), or M2+M3-(C-term:266-296) (D) or with IL-2Ralpha (E) or the IL-2Ralpha /M3 fusion protein (F) were stained with anti-FLAG antibody (A-D) or with anti-IL-2Ralpha antibody (E-F) as described under "Experimental Procedures." x-y (upper panels; projected z- series) and x-z (lower panels; vertical section taken at the level of the white line) images for each construct are shown. Bar = 15 µm.

One important property of basolateral sorting signals is that they are autonomous, i.e. they can confer basolateral targeting to an unrelated, heterologous protein. To test whether the M3 basolateral sorting signal acts in an autonomous fashion, we added M3 Ala266-Gln296 to the C terminus of IL-2Ralpha (Tac antigen), a single-pass transmembrane protein with a short cytoplasmic tail (35). Consistent with previous results (13), wild-type IL-2Ralpha had a predominantly apical distribution when expressed in MDCK cells (Fig. 5E), similar to wild-type M2 (Fig. 5A). By contrast, a substantial fraction of the IL-2Ralpha /M3 fusion protein was found in the basolateral domain, with strong immunoreactivity in the lateral subdomain, although a significant portion of the fusion protein was still detected apically (Fig. 5F). Thus, the M3 basolateral sorting signal can at least partially redirect IL-2Ralpha to the basolateral domain, suggesting that it can confer basolateral targeting to a heterologous protein.

The above results demonstrate that a 31-amino acid sequence, Ala266-Gln296, from the N-terminal portion of the M3 i3 loop contains a basolateral sorting signal in MDCK cells. In an attempt to further define the basolateral targeting determinant, we created M2+M3 fusion proteins in which shorter segments of M3 sequence were fused to the C-terminal coding residue of M2 to investigate whether basolateral targeting is lost upon removal of critical amino acids. Analysis of the steady-state distributions of these fusion proteins revealed that addition of just 21 residues of the M3 i3 loop to the C terminus of M2 could still redirect it to the basolateral domain (Fig. 6). Three fusion proteins, M2+M3-(C-term:271-296), M2+M3-(C-term:266-291), and M2+M3-(C-term:271-291), showed a primarily basolateral localization (Fig. 6, D-F) similar to the wild-type M3 receptor (Fig. 6C). To exclude the possibility that addition of any 21-amino acid sequence to the C terminus of M2 would disrupt its apical targeting and result in a basolateral distribution, M3 Lys570-Leu590 was fused to the C-terminal coding residue of M2. This sequence comprises the C-terminal 21 amino acids of M3, which did not alter the apical sorting of M2 when included in a substitution construct (Fig. 3D). M2+M3-(C-term:570-590) exhibited an apical distribution very similar to wild-type M2 (Fig. 6, B and G), indicating that addition of a random 21-amino acid sequence does not disrupt the apical targeting of M2. These data show that a 21-amino acid peptide, Ser271-Ser291 from the i3 loop of M3, provides a basolateral sorting signal capable of rerouting the otherwise apical M2 receptor.


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Fig. 6.   Localization of M2+M3 C-terminal fusion proteins in MDCK cells. A, presented is a schematic diagram showing M3 i3 loop Ala266-Gln296 in single-letter code. Lines and numbers represent sequences and specific residues, respectively, of M3 that were fused to the C-terminal cytoplasmic tail of M2 to create the fusion proteins shown in D-F. BL, basolateral. B-G, MDCK cells transfected with constructs encoding FLAG-tagged M2, M3, or M2+M3 C-terminal fusion proteins were stained with anti-FLAG as described under "Experimental Procedures." x-y (upper panels; projected Z-series) and x-z (lower panels; vertical section taken at the level of the white line) images for each construct are shown. B, M2; C, M3; D, M2+M3-(C-term:271-296); E, M2+M3-(C-term:266-291); F, M2+M3-(C-term:271-291); G, M2+M3-(C-term:570-590). Bar = 15 µm.

Quantitation of mAChR Apical/Basolateral Distributions-- To confirm the basolateral targeting results described above, quantitation of the immunofluorescence signals for selected mAChR constructs in the apical and basolateral domains of MDCK cells was performed using NIH Image. As shown in Fig. 7, ~80% of M3 receptor immunoreactivity was found in the basolateral domain, similar to the value obtained for the basolateral marker beta -catenin (85%), whereas only 30% of M2 immunoreactivity was basolateral, reflecting the predominantly apical distribution of M2. All M2/M3 chimeras that contain either a substitution or addition of the M3 basolateral sorting signal, Ser271-Ser291, displayed a basolateral enrichment of at least 70%, confirming the ability of the sorting signal to redirect M2 to the basolateral domain. In contrast, M2/M3 chimeras that lack these amino acids showed ~30% basolateral immunoreactivity, similar to wild-type M2. Additionally, staining for wild-type IL-2Ralpha was only ~30% basolateral, whereas that for the IL-2Ralpha /M3 fusion protein was 57% basolateral. This result confirms that the M3 basolateral targeting determinant provides a basolateral sorting signal to IL-2Ralpha , although in this case, the basolateral targeting activity is not sufficient to fully override the endogenous apical signals. These data support the immunocytochemical results discussed above and confirm the ability of the M3 basolateral sorting signal to cause significant targeting of both M2 and IL-2Ralpha to the basolateral domain of MDCK cells.


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Fig. 7.   Quantitation of the steady-state distributions of mAChR constructs in MDCK cells. Quantitation of the immunofluorescence signals for selected mAChR constructs and beta -catenin in the apical and basolateral domains of MDCK cells was performed using NIH Image as described under "Experimental Procedures." Data represent the mean fluorescence intensity per unit area in the basolateral domain and are expressed as the percentage of total fluorescence intensity in the apical and basolateral domains. Data are plotted as the mean of two or the mean ± S.E. of three to seven images for each construct.

Functional Analysis of M2/M3 Chimeras-- The sequence containing the M3 basolateral sorting signal overlaps a region of the i3 loop (Arg252-Thr272 of rat M3) shown to be important for functional coupling to the Gq family of G proteins (45, 46). Therefore, we tested whether addition or substitution of the M3 basolateral sorting signal into M2 would either confer M3-like coupling to Gq or interfere with M2 coupling to the Gi family of G proteins. Coupling of mAChRs to the Gq family of G proteins was assessed by examining their ability to activate phospholipase C in response to the muscarinic agonist carbamylcholine (carbachol). In COS-7 cells transfected with the M3 mAChR, treatment with a maximal concentration of carbachol (1 mM) led to an ~2-fold stimulation of phospholipase C activity relative to untreated controls, whereas carbachol treatment of M2-transfected cells resulted in only a 1.2-fold increase in phospholipase C activity (2.36 ± 0.21- and 1.24 ± 0.06-fold stimulation of phospholipase C activity normalized for receptor expression for M3 and M2, respectively; mean ± S.E., n = 4). Three M2/M3 chimeric receptors were tested: M2/M3-(266-296) (substitution construct), M2+M3-(i3:266-296), and M2+M3-(C-term:266-296) (addition constructs to either the i3 loop or the C-terminal tail of M2, respectively). None of these chimeras stimulated phospholipase C activity to a significant extent following treatment with 1 mM carbachol (1.05 ± 0.02-, 1.14 ± 0.07-, and 0.99 ± 0.04-fold stimulation for M2/M3-(266-296), M2+M3-(i3:266-296), and M2+M3-(C-term:266-296), respectively; mean ± S.E., n = 4). These data indicate that the M3 basolateral sorting determinant is not sufficient to confer Gq coupling to the M2 mAChR.

M2 receptor coupling to the Gi family of G proteins was assessed in JEG-3 human choriocarcinoma cells by determination of the carbachol-mediated regulation of expression of a luciferase reporter gene under the transcriptional control of a promoter containing a cAMP response element (CRE-luciferase). This system has been used extensively to measure M2- and M4-mediated inhibition of forskolin-stimulated adenylyl cyclase activity and cAMP production (33, 34, 37, 41, 47). Consistent with previous results (33, 34, 41), the M2 receptor showed a concentration-dependent inhibition of forskolin-stimulated CRE-luciferase activity (Fig. 8). In contrast, the M3 mAChR showed stimulation of CRE-luciferase activity (Fig. 8), presumably due to the inability of M3 to couple to Gi and to ectopic coupling of M3 to Gs. Similar results have been observed previously for the M1 receptor in this system (33, 41). All three chimeric receptors tested inhibited forskolin-stimulated CRE-luciferase activity in a concentration-dependent manner (Fig. 8). Whereas both substitution and addition of M3 sequence to the M2 i3 loop resulted in inhibition of CRE-luciferase activity to a similar extent as wild-type M2 (51 ± 4, 57 ± 4, and 58 ± 4% inhibition by 10-5 M carbachol for M2, M2/M3-(266-296), and M2+M3-(i3:266-296), respectively; mean ± S.E., n = 4), addition of M3 sequence to the C terminus of M2 (M2+M3-(C-term:266-296)) resulted in 35 ± 6% inhibition by 10-5 M carbachol (mean ± S.E., n = 4). This difference could be due either to reduced functional coupling or to lower cell-surface expression of the fusion protein compared with wild-type M2. To distinguish between these possibilities, we determined the level of each receptor at the cell surface using N-[3H]methylscopolamine, a membrane-impermeable muscarinic antagonist. Under the same transfection conditions used in the functional assay, the M2/M3 chimeras were expressed at similar levels (358 ± 17, 381 ± 2, and 356 ± 52 fmol/mg protein for M2/M3-(266-296), M2+M3-(i3:266-296), and M2+M3-(C-term:266-296), respectively; mean ± S.E., n = 3), which were slightly higher than those for wild-type M2 and M3 (215 ± 41 and 261 ± 30 fmol/mg protein, respectively; mean ± S.E., n = 3). Therefore, the slightly reduced ability of the M2+M3-(C-term:266-296) receptor to inhibit forskolin-stimulated CRE-luciferase activity is likely due to a slightly reduced efficiency of coupling to Galpha i2. Taken together, the data suggest that the M2/M3 chimeric receptors fold correctly, reach the cell surface, and stimulate a functional response similar to the wild-type M2 mAChR. Thus, addition or substitution of the M3 basolateral sorting signal does not appear to substantially alter M2 receptor conformation or function.


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Fig. 8.   Inhibition of forskolin-stimulated adenylyl cyclase activity by M2/M3 chimeric receptors in JEG-3 cells. JEG-3 cells were transfected with constructs encoding the M2 (black-square), M3 (), M2/M3-(266-296) (black-triangle), M2+M3-(i3:266-296) (), or M2+M3-(C-term:266-296) (open circle ) receptor and treated with the indicated concentrations of carbachol as described under "Experimental Procedures." Data are expressed as the percentage of forskolin-stimulated luciferase activity measured in the absence of carbachol and represent the mean ± S.E. of four experiments performed in triplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES

The goal of this study was to characterize the molecular mechanisms and signals involved in the polarized targeting of mAChR subtypes. Examination of the steady-state distributions of mAChRs in MDCK cells revealed that the M2 and M3 receptors are targeted to opposite domains (Fig. 1). This is the first demonstration of differential sorting of highly homologous members of a single G protein-coupled receptor family in MDCK cells. We utilized a gain-of-function approach to identify a basolateral sorting signal in the M3 receptor by analysis of M2/M3 chimeric receptor constructs. The use of chimeras between closely related proteins with opposite phenotypes is advantageous over studies using deletion or truncation mutagenesis because it greatly reduces the possibility that a loss of receptor targeting is due to a generalized effect on protein structure. This consideration is especially important for polytopic membrane proteins such as G protein-coupled receptors.

The M3 basolateral sorting signal is contained within a 21-amino acid sequence, Ser271-Ser291, from the N-terminal portion of the M3 i3 loop. Addition of this signal to the M2 receptor in either the i3 loop or the C-terminal tail caused M2 to be redirected from the apical domain to the basolateral domain of MDCK cells, whereas addition of an irrelevant 21-residue sequence did not alter the apical distribution of M2. Furthermore, substitution of a 70-amino acid region of M3 containing the basolateral determinant also conferred basolateral targeting to the otherwise non-polarized M1 mAChR.3 Together, the data show that M3 Ser271-Ser291 contains a basolateral sorting signal that acts in a position-independent manner and is dominant over targeting signals in other mAChR subtypes. Interestingly, this sequence conferred partial basolateral targeting when transferred to the apical IL-2Ralpha ; although a substantial fraction of the chimeric molecules were redirected to the basolateral domain, a detectable fraction remained apical. The incomplete basolateral targeting of IL-2Ralpha by the M3 basolateral sorting signal suggests that the targeting activity of this basolateral determinant is not sufficient to completely override the activity of the apical signals in IL-2Ralpha , although it can completely counteract the apical signals in M2. Thus, the apical targeting information in IL-2Ralpha may be stronger than that in M2, perhaps due either to higher signal strength or to a greater number of apical targeting determinants. This notion is consistent with recent suggestions that basolateral sorting signals may not always be dominant over apical signals and that the overall targeting phenotype of a protein may be determined by the relative strength (9) or valence (48) of multiple sorting signals. Alternatively, we cannot rule out the possibility that incomplete basolateral targeting of the IL-2Ralpha /M3 fusion protein is due to saturation of the basolateral targeting pathway and spillover of excess protein into the apical pathway, perhaps resulting from higher expression of IL-2Ralpha /M3 as compared with the M2/M3 chimeras.

The M3 basolateral sorting signal identified in this study is sufficient to confer basolateral targeting to other mAChR subtypes. However, an M3 deletion mutant lacking Ala266-Gln296 displayed a basolateral distribution virtually identical to that of the wild-type M3 receptor.2 Thus, the Ser271-Ser291 domain is not the only region of M3 that can mediate its basolateral targeting. Our results suggest that the sequence identified here is the strongest in terms of conferring basolateral targeting activity to heterologous proteins, but other elements of the M3 receptor may mediate its basolateral sorting in the absence of this signal. These other M3 basolateral sorting elements may not be strong enough to counteract the apical signals in M2 and so would not be detected in our experimental approach, but may mediate basolateral targeting of M3 in the absence of any additional signals. Consistent with this idea, it has been reported that the polarized sorting of other proteins can be mediated by multiple, independent targeting motifs (8, 10, 49).

One common feature of basolateral sorting signals is that they sometimes overlap with sequences involved in endocytosis from the plasma membrane (10-12, 44, 50). However, the M3 basolateral sorting signal, which resides in the N-terminal portion of the i3 loop, does not coincide with known internalization motifs for M3 or the highly homologous M1 receptor, which are located in the middle and the immediate membrane-proximal portions of the loop (51, 52). Targeting studies of other proteins have also revealed that basolateral sorting signals can be spatially distinct from endocytosis signals (4, 13, 27, 53, 54).

The M3 basolateral sorting signal has a 3-amino acid overlap (Ser271-Thr273) with a membrane-proximal region of the i3 loop implicated in M3 receptor coupling to the phospholipase C pathway via the Gq family of G proteins (45, 46). For this reason, we tested whether the basolateral targeting motif could also confer coupling to phospholipase C. M2/M3 chimeras containing the basolateral sorting signal did not stimulate phosphatidylinositol turnover, demonstrating that it is not sufficient for coupling to Gq proteins. This is not surprising, as there is minimal overlap between the two motifs, and G protein coupling of mAChRs is thought to be mediated by a multisite domain (46). The basolateral targeting motifs for other G protein-coupled receptors are also distinct from their functional G protein-coupling domains (27, 53, 55).

Basolateral targeting signals have now been identified in many transmembrane proteins. Although no consensus sequence exists, structural determinants such as tyrosine-based (10, 27, 49, 50, 56) or dihydrophobic (11-13, 49) motifs are often found in basolateral sorting signals. However, some basolateral targeting sequences act independently of these motifs (7, 54, 57, 58) or do not contain them at all (4, 59). The M3 basolateral sorting signal (Ser271-Ser291) does not contain a critical tyrosine or a dihydrophobic motif. Thus, the sequence identified here may represent a novel basolateral targeting determinant. Either the continuous amino acid sequence itself or a three-dimensional epitope formed by noncontiguous elements within the sequence may form the actual signal for basolateral targeting.

Heterologous protein expression studies have suggested that epithelial cells and neurons use common cellular mechanisms to generate a polarized distribution of membrane proteins. Based on the sorting of viral glycoproteins, it was initially proposed that the apical domain of epithelial cells corresponds to neuronal axons, whereas the basolateral domain corresponds to cell bodies and dendrites (60). Although this parallel does not hold true for all proteins (61, 62), recent studies have confirmed that some basolateral proteins in MDCK cells are restricted to the somatodendritic domain of cultured hippocampal neurons and that the same signals used for basolateral targeting are also likely to mediate somatodendritic targeting (63). However, proteins that are apical in MDCK cells are not restricted to the axon, but instead are distributed uniformly throughout the axon and dendrites of cultured hippocampal neurons (63). These and other studies have suggested the existence of "axon-including" signals, rather than signals that mediate targeting to the axon exclusively (64). The differential targeting of the M2 and M3 mAChRs in MDCK cells (Fig. 1) and neurons in vivo (24, 25) suggests that similar signals may operate to achieve polarized sorting of mAChRs in epithelial cells and neurons. While the basolateral sorting signals in M3 may mediate its localization to the somatodendritic domain, the apical targeting information in M2 may allow its inclusion in axons. It will be of interest to determine whether the signals that mediate mAChR targeting in MDCK cells are also important for the differential localization of mAChR subtypes in neurons.

In conclusion, we have identified a novel 21-amino acid sequence from the N-terminal portion of the M3 mAChR i3 loop that mediates basolateral targeting in MDCK cells. This determinant, although not uniquely necessary for the basolateral targeting of M3, is dominant over apical sorting signals in the M2 mAChR and can be transferred to a heterologous protein, IL-2Ralpha , in an autonomous fashion. The findings reported here add significantly to our knowledge of the signals that underlie the polarized targeting of G protein-coupled receptors. Future work will be aimed at further elucidation of the molecular signals and cellular machinery involved in mAChR sorting in both epithelial cells and neurons.

    ACKNOWLEDGEMENTS

We thank Dr. Keith Mostov for the gift of MDCK cells, Dr. Warren J. Leonard for the gift of pIL2R3 cDNA, Paulette Brunner (Keck Center for Advanced Studies in Neural Signaling, University of Washington) for assistance with confocal microscopy, and Dr. Michael Schlador and Renee Chmelar for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant NS26920 (to N. M. N.) and by a postdoctoral fellowship in pharmacology/morphology from the Pharmaceutical Research and Manufacturers of America Foundation (to L. S. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, P. O. Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso@u.washington.edu.

Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M007190200

2 L. S. Nadler and N. M. Nathanson, unpublished observations.

3 H. A. Iverson, L. S. Nadler, and N. M. Nathanson, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MDCK, Madin-Darby canine kidney; mAChR, muscarinic acetylcholine receptor; i3 loop, third intracellular loop; PCR, polymerase chain reaction; nt, nucleotides; IL-2Ralpha , interleukin-2 receptor alpha -chain; PBS, phosphate-buffered saline; CRE, cAMP response element; TM, transmembrane domain.

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ABSTRACT
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DISCUSSION
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