mLin-7 is localized to the basolateral surface of renal epithelia via its NH2 terminus

Samuel W. Straight1, David Karnak2, Jean-Paul Borg3, Emmanuel Kamberov3, Heidi Dare3, Ben Margolis1,2,3, and James B. Wade4

3 Howard Hughes Medical Institute, Departments of 1 Internal Medicine and 2 Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109; and 4 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1559


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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In Caenorhabditis elegans, the basolateral localization of the Let-23 growth factor receptor tyrosine kinase requires the expression of three genes: lin-2, lin-7, and lin-10. Mammalian homologs of these three genes have been identified, and a complex of their protein products exists in mammalian neurons. In this paper, we examine the interaction of these mammalian proteins in renal epithelia. Coprecipitation experiments demonstrated that mLin-2/CASK binds to mLin-7, and immunofluorescent labeling showed that these proteins colocalized at the basolateral surface of Madin-Darby canine kidney cells and renal epithelia. Although labeling intensity varied markedly among different renal epithelial cells, those cells strongly expressing mLin-7 also showed intense mLin-2/CASK labeling. We have also demonstrated that mLin-2/CASK binding requires amino acids 12-32 of mLin-7 and have shown that this region of mLin-7 is also necessary for the targeting of mLin-7 to the basolateral surface. Furthermore, the overexpression of mLin-2/CASK mutants in Madin-Darby canine kidney cells caused endogenous mLin-7 to mislocalize. In summary, the NH2 terminus of mLin-7 is crucial for its basolateral localization, likely through its interaction with mLin-2/CASK.

protein targeting; Madin-Darby canine kidney cells; kidney; protein interactions; epithelia


    INTRODUCTION
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INTRODUCTION
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MANY CELLS HAVE POLARIZED plasma membrane compartments. Epithelia are polarized into apical and basolateral plasma membrane domains separated by tight junctions (9). Similarly, neurons are polarized to form the dendrite and axonal components (11). Several mechanisms appear to be involved in the proper distribution of proteins to basolateral or apical surfaces. These mechanisms include the sorting of proteins from the trans-Golgi network and endocytic compartments to the apical or basolateral surface and selective retention of proteins at basolateral or apical membranes (8, 24).

The machinery in cells that recognizes these basolateral-targeting signals and effects targeting of proteins is poorly understood. However, genetic systems are now beginning to reveal the mechanisms that appear important in this targeting process at the plasma membrane (12, 21). Kim and colleagues (21) recently identified a mechanism for basolateral targeting in Caenorhabditis elegans. This mechanism involves three proteins, Lin-2, Lin-7, and Lin-10, that are required for the basolateral targeting of receptors in worm epithelia and neurons (18, 29). Mutations in lin-2, lin-7, or lin-10 lead to a failure of vulva formation, presumably due to mislocalization of Let-23 (16, 18, 31). Lin-2 is a member of the family of proteins known as membrane-associated guanylate kinases (MAGUKs). These proteins contain a region similar to guanylate kinase, an enzyme that converts GMP to GTP. In MAGUKs, however, this domain appears to be catalytically inactive and functions in protein interactions (19, 23, 32). Members of this family also contain PDZ domains, named for three MAGUK proteins, <UNL>P</UNL>SD-95, <UNL>D</UNL>iscs Large, and <UNL>Z</UNL>ona Occludens-1. PDZ domains bind the extreme COOH terminus of certain proteins, and are best understood for their role in the ability of MAGUK-family proteins, such as PSD-95, to bind and cluster ion channels and receptors in synapses (13, 22, 30). The Zona Occludens proteins are MAGUK proteins localized at tight junctions, but the role of the PDZ domains in these proteins is still under investigation (14). The Lin-10 protein contains two PDZ domains and one phosphotyrosine binding (PTB) domain (18). PTB domains were originally identified for their role in phosphotyrosine-dependent interactions in proteins such as Shc and IRS-1. However, recent studies show that they also have an important role in binding beta-turn peptides independent of phosphotyrosine (2). Lin-7 is a smaller protein with a single PDZ domain that binds to the COOH-terminal tail of the Let-23 protein, a receptor tyrosine kinase related to the mammalian epidermal growth factor receptor family (31).

Recent work has indicated that mammalian homologs of the Lin-2, Lin-7, and Lin-10 proteins are present in mammalian neurons in a stable protein complex (4, 7). Mammalian Lin-10 homologs have been previously identified in mammalian cells as the X11 family of proteins (3). They have been also been described as Munc-18 interacting (Mint) proteins (26). There are at least three forms of X11 in mammals that have divergent NH2 termini. The mammalian Lin-2 protein (mLin-2) has been identified in mammalian cells as a protein known as CASK (15), and the NH2 terminus of X11alpha (Mint1) binds to the calmodulin kinase-like domain in mLin-2/CASK (4, 7). The NH2 terminus of mammalian Lin-7 (mLin-7) binds to mLin-2/CASK (4, 7, 18). The X11, mLin-2/CASK and mLin-7 proteins have not been extensively studied in mammalian epithelial cells, although mLin-2/CASK has been found at the basolateral surface of epithelia (10). Therefore, we sought to examine the interactions of mLin-7, mLin-2/CASK, and X11 in MDCK and renal epithelial cells. We have found that mLin-2/CASK and mLin-7 interact in renal epithelial cells, but they do not interact with X11gamma , the form of X11 we have identified in epithelial cells. Both mLin-2/CASK and mLin-7 are located at the basolateral surface of epithelial cells. In renal epithelia, there is close correlation in the expression of mLin-7 and mLin-2/CASK in different tubule segments. Finally, we have further refined the region of mLin-7 required for interaction with mLin-2/CASK and determined that the region of mLin-7 that mediates its interaction with mLin-2/CASK is also required for its basolateral targeting.


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DNA constructs. The cloning of full-length X11alpha and X11gamma cDNAs, as well as the human lin-2 and mouse mlin-7 cDNAs, has been described previously (4, 5). The lin-2 and mlin-7 cDNAs were used as templates for PCR with appropriate oligonucleotide primers to create truncated protein constructs. The cDNAs or PCR products were subcloned into the plasmid pGSTag by using appropriate restriction endonucleases, where GST denotes glutathione-S-transferase. This plasmid and the expression and purification of GST-fusion proteins have been described previously (3). The mlin-7 cDNA was subcloned into the plasmid pET28a+ (Novagen; Madison, WI) for the creation of a His6-tagged protein. The vectors pRK5 and pRK5-Myc (3) were used to express proteins in mammalian cells with or without an NH2-terminal Myc epitope, respectively. Canine betaine-gamma amino butyric acid transporter-1 (BGT-1) cDNA (34), obtained from Joseph Handler and H. Moo Kwon, was subcloned into pRK5-Myc. All constructs were sequenced by using Sequenase version 2.0 DNA sequencing kit (Amersham Life Science, Cleveland, OH) or by automated sequencing at the University of Michigan DNA Sequencing Core.

Cell culture and transfection. Human embryonic kidney 293T (HEK293T) and Madin-Darby canine kidney (MDCK) cells were grown in DMEM (Life Technologies, Grand Island, NY) containing 100 U/ml penicillin and 100 µg/ml streptomycin sulfate, supplemented with 10% FCS.

HEK293T were transiently transfected by using a calcium phosphate precipitation method. MDCK cells were stably transfected by using FuGENE 6 transfection reagent (Boehringer Mannheim, Mannheim, Germany) followed by selection with Geneticin/G-418 (600 µg/ml active; Life Technologies) or hygromycin B (500 µg/ml; Invitrogen, Carlsbad, CA) depending on the selectable marker plasmid initially cotransfected. Studies were conducted with both pools of selected cells and with established clonal cell lines.

Antibodies. Polyclonal anti-mLin-7, anti-mLin-2/CASK, and anti-X11 antibodies used for immunoprecipitation, immunoblotting, and immunostaining were prepared by injecting rabbits with purified GST-fusion proteins as follows: GST-mLin-7, GST-mLin-2/CASK (1-275), GST-mLin-2/CASK (578-897), GST-X11alpha (620-837), and GST-X11gamma (15-246).

The anti-mLin-7 antibody was subsequently affinity purified and was used in all applications. For purification, His6mLin-7 was expressed in DE3 Escherichia coli, which were lysed, and the His-tagged protein was bound to nickel-agarose beads (Qiagen, Santa Clara, CA) and purified. The bound protein was eluted with 0.5 M imidazole, dialyzed, and then coupled to Affigel-10 (Bio-Rad Laboratories, Hercules, CA). Anti-mLin-7 antiserum was precipitated with 50% ammonium sulfate, and the precipitated proteins were resuspended and dialyzed against PBS. The antibody solution was passed several times over an Affigel-His6mLin-7 column, and the bound antibody was eluted under acid (pH 2.5) conditions. The His6mLin-7 protein was also used in immunofluorescence applications to competitively inhibit immunostaining with anti-mLin-7 antibody.

Anti-Myc monoclonal antibody (clone 9E10) was used for immunostaining and immunoblotting. The anti-mLin-2/CASK monoclonal antibodies used for immunolocalization in the kidney were from Transduction Laboratories (Lexington, KY). Affinity-purified rabbit polyclonal anti-ZO-1 antibodies and rat anti-uvomorulin/E-cadherin monoclonal antibodies used for immunostaining were purchased from Zymed (San Francisco, CA) and Sigma Chemical (St. Louis, MO), respectively. Unless otherwise noted, fluorochrome-conjugated secondary antibodies used in immunostaining procedures were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Antibodies to COOH-terminal peptides of aquaporin-2 (AQP2; LC54) and -3 (AQP3; LP45) were raised in chickens to peptides previously utilized for production of these antibodies in rabbits (33). Rabbit antibody to NKCC2 (L320) was raised to amino acids 33-55 of the rat cotransporter (20) and was kindly provided by Dr. Mark Knepper.

Immunostaining of MDCK cells. For immunostaining procedures, MDCK cells were seeded at high density onto acid-washed coverglasses or Transwell PTFE membrane filters (0.4 µm pore size; Corning Costar, Cambridge, MA). The cells were allowed to grow to confluence to form a polarized monolayer. After being washed with PBS, the cells were fixed with 4% formaldehyde/PBS and permeabilized with 0.1% Triton X-100/PBS. After being blocked for 1 h with goat serum, the cells were incubated with primary antibodies diluted in 2% goat serum/PBS in a humidified chamber for 1 h (affinity-purified anti-mLin-7 at 1:50; anti-Myc at 1:400; anti-ZO-1 at 1:400; and anti-E-cadherin at 1:1,600). After being washed three times with 2% goat serum/PBS, the cells were incubated with secondary antibodies coupled to FITC, Cy3, or Cy5 (diluted at 1:500 in 2% goat serum/PBS) for 1 h in a humidified chamber. Coverglasses were mounted on glass slides with ProLong antifade reagent (Molecular Probes, Eugene, OR). Membrane filters were cut from their plastic casing with a scalpel and mounted as above. Examination of immunostained cells was performed on an Olympus BX60 fluorescent microscope, and digital images were taken with a SPOT charge-coupled device camera (Diagnostic Instruments). Confocal laser-scanning microscopy was performed on a Nikon Diaphot 200 microscope paired with a Noran laser and InterVision software (Noran Instruments, Middleton, WI) at the Morphology and Image Analysis Core of the University of Michigan Diabetes Research Center.

Protein procedures. Lysates for precipitation experiments were prepared from subconfluent HEK293T and MDCK cells in 10- or 15-cm tissue culture dishes, respectively (3). Cells were washed twice with cold PBS and lysed in 0.5-1 ml of lysis buffer [50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA] supplemented with 1 mM phenylmethylsulfonylfluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, 10 mM Na4P2O7, and 200 µM Na3VO4. The lysates were cleared by centrifugation at 16,000 g for 20 min at 4°C to remove insoluble debris. For precipitation assays, 0.2 ml lysate from transiently transfected HEK293T or 1 ml lysate from MDCK cells was used.

Fractionated lysates were collected from harvested mouse kidneys snap-frozen in liquid nitrogen. The frozen organ was ground by mortar and pestle in hypotonic buffer [10 mM Tris · Cl (pH 7.4), 0.2 mM MgCl2, 5 mM KCl] supplemented with 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (7.5 ml buffer/0.5 g organ). The organ was then homogenized by 20 strokes in an ice-cold dounce (B piston). Sucrose was added to the homogenate to a final concentration of 0.25 mM, and EDTA to 1 mM, and nonhomogenized debris was removed by centrifugation at 1,000 g for 10 minutes at 4°C. The supernatant was then centrifuged at 138,000 g (max) for 1 h at 4°C. The supernatant was collected and was termed the cytoplasmic fraction. The pellet was resuspended in a volume of lysis buffer (described above) equivalent to the volume of the cytoplasmic fraction. After 30 min on ice, insoluble debris was removed by centrifugation at 16,000 g for 30 min at 4°C. The resulting supernatant was termed the membrane fraction. Typically, 1 ml of fractionated lysate was used in precipitation assays.

For immunoprecipitation, lysates were incubated with antibodies overnight at 4oC. Protein A-agarose was added and immune complexes bound to beads were recovered after 1 h, washed three times with HNTG buffer (50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 0.1% Triton X-100), boiled in 1× sample buffer, and separated by SDS-PAGE. Transfer and immunoblotting on nitrocellulose using HRP-protein A or HRP-anti-mouse antibody were performed as described (3) by using the Renaissance chemiluminescence reagent (NEN Life Science Products, Boston, MA).

GST-fusion protein production and GST-binding assays were performed as previously described (3). Typically, 5 µg of GST-fusion protein were used per precipitation reaction.

Immunolocalization in the rat kidney. Renal tissue was obtained from 180- to 250-g male Sprague-Dawley rats fixed by perfusion via the abdominal aorta. Perfusion was for 2 min in PBS to clear the kidneys of blood, 5 min in 2% paraformaldehyde, and 2 min in a cryoprotectant of 10% EDTA in 0.1 M Tris. Fixed kidneys were sliced and further incubated in the cryoprotectant for 60 min, wrapped in aluminum foil, and frozen on dry ice. Cryostat sections 12-15 µm thick were made and picked up on coverslips coated with HistoGrip (Zymed). Sections were usually further fixed and attached to the coverslips by incubation for 5 min with 4% paraformaldehyde and then treated with either 1% SDS or 6 M guanidine for 10 min to unmask antigenic sites (6). Sections were then washed three times with high-salt buffer (50 ml PBS, 0.5 g BSA, 1.13 g NaCl), incubated in blocking agent (50 ml PBS, 0.5 g BSA, 0.188 g glycine, pH 7.2) for 20 min, followed by incubation with primary antibody overnight at 4°C. Primary antibodies were diluted to 10 µg/ml with incubation medium (50 ml PBS, 0.05 g BSA, 200 µl 5% NaN3). After this incubation, sections were rinsed five times with high-salt buffer before incubation with secondary antibody for 2 h at 4°C. Appropriate species-specific antibodies coupled to Alexa 488 or 568 dyes (Molecular Probes) were diluted 1:200 with incubation medium. These samples were again washed five times with high-salt buffer over the course of 1 h and then in PBS to remove the excess salt before mounting and confocal microscopy.


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The structures and binding sites for X11alpha , mLin-2/CASK, and mLin-7 are shown in Fig. 1A. The form of mLin-7 utilized in these studies corresponds to the protein Veli-3 described by Butz et al. (7). The calmodulin kinase II-like (CKII) domain of mLin-2/CASK binds to a region within the NH2 terminus of X11alpha , whereas the NH2 terminus of mLin-7 binds the region between the CKII and PDZ domain of mLin-2/CASK (1, 4, 7, 18). We have previously generated antibodies that recognize mammalian mLin-2/CASK and mLin-7 (4). In MDCK cells, we confirmed that mLin-2/CASK was bound to mLin-7 (Fig. 1B). Both mLin-2/CASK and mLin-7 were detected in the cytosol and membrane fractions of mouse kidney lysates in approximately equal quantities (results not shown), and in both compartments mLin-2/CASK and mLin-7 were bound to each other. We have previously detected X11gamma as an epithelial form of X11 in mammalian cells. Unlike mLin-7 and mLin-2/CASK, most of X11gamma is present in the cytosolic fraction of kidney lysate (Fig. 2A). When we immunoprecipitated with antibodies to mLin-7, we detected no X11gamma in the mLin-2/CASK-mLin-7 complex of kidney. Similarly, antibodies to X11gamma did not coimmunoprecipitate mLin-2/CASK or mLin-7. In MDCK cells, the expression of X11gamma is relatively low, and we could not detect mLin-2/CASK or mLin-7 in a complex with X11gamma (data not shown). To better test these interactions in tissue culture cells, we overexpressed X11gamma or X11alpha in MDCK cells. There is no endogenous X11alpha in MDCK cells, but it was easily detected when overexpressed (Fig. 2B). As expected, mLin-2/CASK and mLin-7 were found to coimmunoprecipitate with X11alpha in these cells when both anti-X11alpha and anti-mLin-7 were used as immunoprecipitating antibodies. In contrast, X11gamma did not coimmunoprecipitate with mLin-2/CASK or mLin-7 even after X11gamma was overexpressed in MDCK cells (Fig. 2C). The divergence in the amino acid sequences of their NH2 termini, particularly in the region of X11alpha that binds to mLin-2/CASK, might explain the differential inclusion of X11alpha and X11gamma in a complex with mLin-2/CASK and mLin-7 (5). One final observation was that X11gamma appears as three immunoreactive bands in Fig. 2, A and C. These multiple bands are not likely to be the product of alternate splicing, because the same bands were observed from both endogenous (Fig. 2A) and exogenous (Fig. 2C) expression. Nor are they likely to be the result of proteolytic degradation during the course of the experiments, because proteases were present in the lysis buffer. More likely, the three immunoreactive bands are the result of a posttranslational modification, such as phosphorylation, but this remains to be determined by future investigation.


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Fig. 1.   Lin-2, Lin-7 and Lin-10 are evolutionarily conserved proteins. A: schematics of mammalian homolog of Lin-2, Lin-7, and Lin-10 proteins of Caenorhabditis elegans. Left: names of proteins; right: relative molecular weights (kDa). Sites of interaction (BS) between these proteins are demarcated by labeled bars above or below each illustration. Recognized protein domains are indicated as follows: PTB, phosphotyrosine binding; PDZ, PSD-95/Dlg-1/ZO-1; CKII, calmodulin kinase II; SH3, Src-homology 3; GK, guanylate kinase. Hook indicates region recognized as binding cytoskeletal protein 4.1 (10). B: coimmunoprecipitation of mLin-2/CASK and mLin-7. mLin-2/CASK and mLin-7 proteins were coprecipitated from Madin-Darby canine kidney (MDCK) cells and from fractionated mouse kidney lysates with affinity-purified anti-mLin-7 antibodies and protein A-Sepharose beads. Preimmune rabbit serum was used as a control. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and identified by immunoblot with antibodies indicated. Left: relevant bands (arrows); right: relative molecular weight. memb, Membrane; cyto, cytoplasm.



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Fig. 2.   X11gamma does not bind mLin-2/CASK or mLin-7. A: interaction of X11gamma , mLin-2/CASK, and mLin-7 in kidney extracts. Mouse kidney cytoplasm and membrane lysate fractions were incubated with antibodies to either mLin-7 or X11gamma or with preimmune rabbit serum as a control. Bound proteins were precipitated with protein A-Sepharose beads, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies indicated. Note that in immunoblot for mLin-2, a nonspecific band of ~100 kDa appears in anti-X11gamma immunoprecipitation lane. B and C: interaction of X11 protein, mLin-2/CASK, and mLin-7 in transfected MDCK cells. Triton-soluble lysates were collected from untransfected MDCK cells (WT) or MDCK cells stably transfected with a plasmid expressing X11alpha (+X11alpha ) as indicated in B (top). Proteins were immunoprecipitated from these cells with antibodies to mLin-7, X11alpha , or with preimmune rabbit serum as a control, and treated as described above. In C, lysates were collected from MDCK cells stably transfected with a plasmid expressing X11gamma . Proteins in these lysates were immunoprecipitated with antibodies to mLin-7, X11gamma , or with preimmune rabbit serum as a control. Precipitated proteins were treated as described above. In all panels, relative molecular weight is indicated to the right (kDa), and arrows to left of immunoblots indicate relevant bands. In B and C, lysate indicates a lane loaded with 1/20 the volume of total cell lysate used for precipitation.

Previous work had indicated that the NH2-terminal region of mLin-7 was responsible for binding to mLin-2/CASK (4, 7, 18). We further refined the region of mLin-7 that binds to mLin-2/CASK by using GST-fusion proteins to several deletion mutants of mLin-7 (shown in Fig. 3A) and were able to show that amino acids 1-43 were sufficient to mediate this interaction (Fig. 3B). Further minimization of the NH2 terminus to amino acids 1-33 of mLin-7 resulted in greatly reduced binding to mLin-2/CASK (data not shown). Removal of the first 12 amino acids of mLin-7 had no effect on the interaction of mLin-2/CASK with mLin-7 while removing the first 32 amino acids, or more, completely eliminated the interaction (Fig. 3C). Altogether, these results indicate that amino acids 12-32 of mLin-7 are required for binding to mLin-2/CASK, although other elements within the NH2 terminus may be necessary for efficient binding.


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Fig. 3.   Deletion mutagenesis of mLin-7. A: schematic shows full-length mLin-7 protein (top) and mLin-7 constructs created for this study (bottom). Nos. to left correspond to amino acids of mLin-7 contained within each construct, whereas nos. on illustrations indicate amino acid sequence position. PDZ, PSD-95/Dlg-1/ZO-1 protein interaction domain. B and C: binding of deletion mutants to mLin-2. Glutathione-S-transferase (GST)-fusion proteins were made with each mLin-7 construct shown in A, bound to glutathione-agarose beads, and used to precipitate Myc-mLin-2/CASK from HEK293T lysates. Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Myc antibodies as indicated. Arrows to left of immunoblots indicate bound Myc-mLin-2/CASK. GST alone is a control construct not containing any mLin-7 sequence. Lane labeled lysate indicates 1/10 amount of HEK293T total lysate used in precipitation experiments.

Next we used our antibodies to examine the localization of mLin-7 in MDCK cells (Fig. 4). Shown are the immunolocalization experiments involving MDCK cells affixed to coverglasses. Comparable results were obtained with cells on PTFE membrane filters (data not shown). Using rabbit polyclonal antibodies to mLin-7, we detected endogenous mLin-7 at the lateral surface of MDCK cells (Fig. 4A). Using anti-Myc antibodies we determined that expressed Myc-mLin-7 was localized to the same site as the endogenous protein (Fig. 4B). Neither our polyclonal antibodies nor commercial antibodies to mLin-2/CASK were suitable for immunofluorescence studies of MDCK cells, so we were unable to determine the location of endogenous mLin-2/CASK. However, we also found Myc-tagged mLin-2/CASK was localized to the lateral surface of MDCK cells (see below, Fig. 9C). In contrast, overexpressed X11gamma localizes to a perinuclear region and not at the cell surface (Fig. 4C). This perinuclear localization of X11gamma corresponds with the localization of X11alpha in neurons (1). Furthermore, a similar perinuclear localization has been observed for X11alpha expressed in MDCK cells (data not shown). These results suggest that some conserved region(s) of the X11 proteins might be responsible for their localization in cells, and that the machinery involved in this localization is conserved in both neurons and epithelial cells.


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Fig. 4.   Localization of mLin-7 and X11gamma in MDCK cells. MDCK cells were seeded onto glass coverslips, fixed, permeabilized, and stained with indicated primary antibodies, followed by staining with secondary antibodies conjugated to fluorochromes. A: endogenous mLin-7 protein in untransfected MDCK cells stained with affinity purified anti-mLin-7 antibodies. B: MDCK cells stably expressing Myc-tagged mLin-7 and stained with anti-Myc antibodies. C: localization of X11gamma in MDCK cells stably transfected with a plasmid expressing full-length X11gamma . Although a confluent monolayer is shown, and was confirmed by staining with anti-ZO-1 antibodies, not all the cells express X11gamma . Cells are shown at higher magnification to show detail of perinuclear staining pattern. D and E: control immunostaining of MDCK cells with anti-ZO-1 antibodies (D) and anti-E-cadherin antibodies (E), which demarcate tight junction and adherens junction respectively. In A-E, top panel shows digital image of immunostained cells acquired through a charge-coupled device camera attached to a fluorescent microscope, and bottom panel shows Z-section of a Z-series taken with a confocal laser-scanning microscope. Bar, 10 µm.

In agreement with our results in MDCK cells, we were also able to localize endogenous mLin-7 and mLin-2/CASK proteins to the basolateral surface of multiple renal segments in rat kidney. Figure 5 shows the smooth localization of mLin-7 at the basolateral surface of inner medullary collecting ducts and, with less intensity, in thin limbs of the loop of Henle (Fig. 5A). The papillary epithelial cells that cover the surface of the renal papilla were also intensely labeled by antibody to mLin-7. The corresponding labeling with antibody to AQP3 is shown in Fig. 5B. This water channel protein is present in the basolateral membrane of collecting ducts and papillary epithelium, but not in thin limbs. Thus mLin-7 has a distinctly basolateral localization in native renal epithelia as well as cultured MDCK cells. Tubular segments in the inner medulla that stained brightly for mLin-7 (Fig. 5C) also stained intensely for mLin-2 (Fig. 5D), demonstrating a close correlation in the expression of the two proteins in native epithelial cells. Similar staining for mLin-2/CASK and mLin-7 at the basolateral surface was also seen in the outer medulla of the kidney (Fig. 6, A and B, respectively). The specificity of the mLin-7 antibody was examined by incubating it with an excess of His6-mLin-7 protein. This blocked labeling by the mLin-7 antibody (Fig. 6C) but not by the mLin-2/CASK antibody (Fig. 6D) and demonstrates that the colocalization of mLin-7 and mLin-2/CASK was not due to cross-reaction of the mLin-2/CASK antibody with shared epitopes present on mLin-7.


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Fig. 5.   Immunolocalization of mLin-7 and mLin-2/CASK in renal inner medulla. A: antibody to mLin-7 shows this protein at basolateral surface of inner medullary collecting ducts (CD). Labeling of lateral aspects is indicated by arrowheads. Thin limbs of loop of Henle (TL) and papillary epithelium (PE) are also labeled. B: labeling with antibody to basolateral water channel aquaporin-3 (AQP3) in collecting ducts and papillary epithelium corresponds closely to mLin-7 labeling. C and D: immunolocalization of mLin-7 (C) compared with labeling by using a mouse monoclonal antibody to mLin-2/CASK (D) demonstrated that these proteins shared a very similar distribution in renal segments. Bar, 25 µm.



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Fig. 6.   Immunolocalization of mLin-7 and mLin-2/CASK in the renal outer medulla. A and B: antibodies to mLin-7 (A) and to mLin-2/CASK (B) label basolateral domains of renal tubules in a similar pattern. Sections labeled with rabbit antibody to mLin-2 and monoclonal mouse anti-mLin-2/CASK demonstrated that both antibodies gave a similar pattern of mLin-2/CASK immunostaining (data not shown). C: incubation of mLin-7 antibody with an excess of His6mLin-7 protein blocked labeling by antibody. D: labeling by mLin-2 antibody was not diminished by preincubation with excess His6mLin-7 protein. Bar, 25 µm.

Immunolocalization of mLin-7 and mLin-2/CASK with respect to segment-specific transporters showed that they are not uniformly expressed along renal nephron segments. Figure 7 illustrates the labeling pattern observed by using antibodies to the Na-K-Cl cotransporter 2 (NKCC2) to identify the apical domains of the epithelial cells in the thick ascending limbs of the loop of Henle (Fig. 7A). Although the basolateral domains of cells comprising the thick ascending limb and collecting duct segments were strongly labeled by mLin-2/CASK and mLin-7 (not shown), proximal tubules were only weakly labeled (Fig. 7B). Close examination of collecting duct labeling showed that the labeling pattern for mLin-7 and mLin-2/CASK varied significantly in intensity, even within a given segment. Labeling with antibodies to the principal cell-specific water channel AQP2 (Fig. 7C) showed that this cell type strongly expressed mLin-7 at the basolateral domain, whereas there was little or no labeling by mLin-7 antibody detected in adjacent intercalated cells (arrows, Fig. 7D).


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Fig. 7.   Immunolocalization of mLin-2/CASK and mLin-7 in renal outer medulla and cortex. A and B: comparison of the labeling with antibody to bumetanide-sensitive cotransporter NKCC2 (A) with respect to mLin-2/CASK labeling (B) shows that many of the brightly labeled tubules in the renal outer medulla and cortex are thick ascending limbs of loop of Henle (TAL). Other structures that are brightly labeled by mLin-2/CASK are CD, whereas proximal tubules (PT) are much more weakly labeled on their basal domains. C and D: comparison of labeling with an antibody to water channel aquaporin-2 (AQP2; C) with respect to mLin-7 (D) shows that mLin-7 expression varies between epithelial cell types even within a segment. Intercalated cells, which are weakly labeled for AQP2 at their apical domains (C, arrows), also show little or no labeling by mLin-7 antibody at their basolateral domains (D, arrows), in contrast to bright staining with both labels seen in adjacent principal cells. Bar, 25 µm.

One potential binding target for mLin-7 in the renal medulla is BGT-1. BGT-1 is expressed in the cells of the inner medulla in response to osmotic stress, localizes to the basolateral surface of cells, and has a COOH-terminal sequence, -EKTHL, remarkably similar to that of worm Let-23, -EKTCL (27, 34). Recent data also suggests that the COOH terminus of BGT-1 was responsible for the retention of the transporter at the basolateral surface of cells (28). Figure 8 shows the coprecipitation of BGT-1 with the PDZ domain of mLin-7: BGT-1 was precipitated with GST-fusion proteins to both full-length mLin-7 and the mLin-7 PDZ domain, GST-mLin-7 (79-197), but not the NH2-terminal half of mLin-7, GST-mLin-7 (1-92). These results taken together support the suggestion that BGT-1 might be targeted to the basolateral surface of medullary collecting duct cells by the mLin-7-mLin-2/CASK complex.


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Fig. 8.   Betaine gamma -amino butyric acid transporter 1 (BTG-1) binds to the PDZ domain of mLin-7. GST-mLin-7-fusion proteins were bound to glutathione-agarose beads, then used to precipitate Myc-BGT-1 from HEK293T lysates. Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Myc antibodies as indicated. Arrow to left of immunoblot indicates Myc-BGT-1, and asterisks indicate higher order (glycosylated) forms of Myc-BGT-1 protein. GST alone is a control construct not containing any mLin-7 sequence. Amino acids of mLin-7 included in fusion proteins are indicated, and these constructs are illustrated in Fig. 3. Lane labeled lysate indicates 1/10 amount of HEK293T total lysate used in precipitation experiments. Relative molecular weight is shown to right (kDa).

In an attempt to dissect the molecular basis for the localization of mLin-7 to the basolateral surface, we expressed the Myc-tagged PDZ (amino acids 79-197) or NH2 terminus (amino acids 1-92) of mLin-7 in MDCK cells. Through the examination of these cells by immunofluorescence, we found that the amino terminus localized to the lateral membrane (Fig. 9A), whereas the PDZ domain of mLin-7 localized diffusely within cells (Fig. 9B). This suggested that the NH2 terminus was responsible for the localization of mLin-7 to the basolateral surface of the cells. Next, we wanted to determine if the mLin-2 binding domain within the NH2 terminus of mLin-7 was essential for mLin-7 localization. Therefore, MDCK cells were transfected with the deletion mutants of mLin-7 shown in Fig. 3. We found that the deletion of the first 12 amino acids of mLin-7 did not alter localization to the basolateral surface of cells (Fig. 9C), whereas deletion of the first 32 amino acids abolished basolateral localization (Fig. 9D). These results indicated that localization of mLin-7 to the basolateral surface of cells depended on the region of mLin-7 also required for binding to mLin-2/CASK. As mentioned previously, we were unable to determine the localization of endogenous mLin-2/CASK in these MDCK cell lines, because our antibodies, as well as those available commercially available, proved to be inadequate for detecting the canine protein by immunofluorescence.


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Fig. 9.   Immunolocalization of mLin-7 constructs expressed in MDCK cells. MDCK cells were stably transfected with Myc-tagged mLin-7 constructs. These cells were prepared for immunostaining as described in Fig. 4. Primary antibody was anti-Myc. As described in Fig. 4, top panel in A-E is a digital photomicrograph, and bottom panel shows a Z-section. A-D show MDCK cells expressing different truncated mLin-7 constructs described in Fig. 3. All panels show confluent monolayers of cells; however, in some cases not all cells express transfected plasmid (B and D). Amino acids contained within each construct are given in parentheses above each panel. Bar, 10 µm.

To assess the role of mLin-2/CASK in mLin-7 localization, we examined the effects of the overexpression of mLin-2/CASK mutants on the localization of endogenous mLin-7. We expressed two Myc-tagged fragments of mLin-2/CASK in MDCK cells: the NH2-terminal half (amino acids 1-612), and the COOH-terminal half (amino acids 578-897). Previous studies have shown that the NH2-terminal half of mLin-2/CASK binds mLin-7, whereas the COOH-terminal fragment does not (4, 7, 18). This was confirmed in the results of experiments involving the coprecipitation of the Myc-tagged mLin-2/CASK constructs with endogenous mLin-7 protein in our MDCK cell lines (Fig. 10A). Examination of these cells by immunofluorescence showed that the Myc-tagged wild-type mLin-2/CASK localizes to the basolateral surface of cells, overlapping in part with the localization of mLin-7 (Fig. 10C, top and bottom panels), as well as with the junctional protein ZO-1 (Fig. 10C, middle panel). However, neither the NH2-terminus nor the COOH terminus of mLin-2/CASK localized to the basolateral surface: the mLin-2/CASK(1-612) localized in a diffusely cytoplasmic pattern (Fig. 10D), whereas mLin-2/CASK(578-897) entered the nucleus (Fig. 10E). The aberrant localization of these halves of mLin-2/CASK indicates the correct localization of mLin-2/CASK to the basolateral surface is likely to be a more complex process than mLin-7 localization, perhaps involving more than one region of the mLin-2/CASK protein. Most interestingly, however, the expression of the NH2-terminal half of mLin-2/CASK was sufficient to mislocalize a significant fraction of mLin-7 away from the lateral membrane (Fig. 10D, top and bottom panels), with no apparent effect on the localization of the junctional protein ZO-1 (Fig. 10D, middle panel). In contrast, overexpression of the COOH-terminal half of mLin-2/CASK had no significant effect on the localization of mLin-7 in cells (Fig. 10E, top and bottom panels), consistent with its inability to bind mLin-7. Thus it appears that in MDCK cells the localization of mLin-7 was dependent on the correct localization of mLin-2/CASK.


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Fig. 10.   Expression and immunolocalization of mLin-2/CASK constructs in MDCK cells. MDCK cells were stably transfected with Myc-tagged mLin-2/CASK constructs. A: coimmunoprecipitation and immunoblotting of transfected mLin-2/CASK constructs with endogenous mLin-7 from MDCK cells by using anti-Myc antibodies, as described in Fig. 1. Antibodies used for blotting are indicated below each panel. Left: relevant bands (arrows); open arrow, immunoglobin heavy chain; right: relative molecular weight (kDa). B: MDCK cells transfected with empty RK5-Myc vector as a control. C: MDCK cells transfected with full-length mLin-2. D and E: MDCK cells expressing truncated mLin-2/CASK proteins. B-E: cells prepared for immunostaining as described in Fig. 4, with anti-Myc, anti-mLin-7 and anti-ZO-1 as primary antibodies. As described in Fig. 4, top panel in A-E is a digital photomicrograph, showing cells colabeled with anti-Myc antibodies (green) and affinity purified anti-mLin-7 antibodies (red); middle panel shows Z-section of a Z-series taken with a confocal laser-scanning microscope of cells stained with anti-Myc (green) and anti-ZO-1 (red) antibodies. ZO-1 is a tight juction-associated protein used as a control to indicate lateral aspect of membrane; and bottom panel also shows a Z-section, but here cells were immunostained with anti-Myc antibodies (green) as well as affinity purified anti-mLin-7 antibodies (red) to show distribution of endogenous mLin-7. In all panels, yellow indicates an overlap in antibody labeling. Amino acids contained within each truncated protein are indicated above each panel in parentheses. Bar, 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In worms, the Lin-7 PDZ domain interacts with the Let-23 growth factor receptor and is believed to be essential for the basolateral localization of the receptor (18, 31). In mammalian epithelial cells, our results have demonstrated that mLin-7 localizes to the basolateral surface of cells. The finding that mLin-7, like mLin-2/CASK, is at the basolateral surface suggests that it may be involved in binding and retaining cell surface proteins at the basolateral membrane, rather than playing a role in Golgi sorting. Many PDZ domain-containing proteins have a role in the clustering of receptors in neurons (22, 30). However, a more detailed examination of mLin-7 and mLin-2/CASK localization by using electron microscopy would be required to determine whether these proteins were clustered at distinct sites on the basolateral plasma membrane of epithelial cells, or more generally localized as the results of our light and confocal microscopy studies indicate. The mLin-7 protein is strongly expressed in certain tubular segments of the kidney, such as the inner medullary collecting duct, where it colocalizes with mLin-2. The PDZ domain of mLin-7 does not appear to bind any members of the mammalian epidermal growth factor receptor family, but it can bind to the COOH terminus of the Let-23 protein (S. W. Straight, J.-P. Borg, D. Karnak, and B. Margolis, unpublished observations). There are proteins within the inner medulla of the kidney that have COOH-terminal sequences similar to worm Let-23, such as BGT-1 (27, 34). The coprecipitation of BGT-1 with the mLin-7 PDZ domain (Fig. 8) suggests that BGT-1 might be targeted to the basolateral surface of kidney epithelial cells by a mLin-7-mediated mechanism. Furthermore, the identification of the COOH-terminal sequence of BGT-1 as a binding partner for the mLin-7 PDZ domain, combined with the knowledge of the COOH-terminal sequence of Let-23, suggests that a further search for proteins with similar COOH-termini may identify other binding partners for the PDZ domain of mLin-7. It should be noted, however, that mLin-7 and mLin-2/CASK are only weakly expressed in some epithelial cell types of the kidney, suggesting this complex may not be operative in all epithelia and is just one of the systems involved in the basolateral localization of proteins.

We have determined that amino acids 1-43 of mLin-7 are sufficient to mediate binding to mLin-2 and have also demonstrated that deletion of amino acids 12-32 of mLin-7 eliminates this interaction with mLin-2. Using the Chou-Fasman method for secondary structure prediction and the determination of Eisenberg hydropathic moment (Lasergene Protean program, DNASTAR), we have identified within this region an amphipathic helix encompassing amino acids 9-27. Our data suggest that this putative amphipathic helix is a crucial component of the mLin-7-mLin-2/CASK interaction. However, a peptide containing amino acids 1-33 of mLin-7 did not significantly block binding of mLin-2/CASK to full-length mLin-7 (S. W. Straight, E. Kamberov, and B. Margolis, unpublished observations), indicating that this region may only comprise part of the necessary elements for efficient binding of mLin-7 to mLin-2/CASK. We also identified the NH2-terminus of mLin-7 as being essential for the localization of mLin-7 in MDCK cells to the basolateral surface. The deletion of amino acids 12-32 prevents mLin-7 from localizing properly to the basolateral surface, as well as negating mLin-2/CASK binding. Furthermore, the overexpression of the NH2-terminal half of mLin-2/CASK pulls a fraction of endogenous mLin-7 from the lateral membrane. Thus our data suggest that the interaction of mLin-7 with mLin-2/CASK is crucial for mLin-7 localization to the basolateral surface of MDCK cells. Further support for this notion comes from the close correlation between mLin-2/CASK and mLin-7 expression in various kidney segments. However, confocal microscopy (Fig. 10) showed only partial overlap in the localization of mLin-2/CASK and mLin-7, indicating that other proteins may be involved in the localization of mLin-7. Butz et al. (7) have shown that DLG2 and DLG3, MAGUK proteins found in neuronal cells, have a region homologous to mLin-2/CASK that binds the NH2 terminus of the Lin-7 homolog Veli-1. It is thus possible that mLin-7 has multiple binding partners that direct its localization in diverse cell types.

Our findings also indicate that mLin-7 and mLin-2/CASK are peripheral membrane proteins that interact both in the cytosol and at the membrane. The processes involved in the targeting of such proteins to basolateral vs. apical surfaces are just beginning to be understood (8, 17, 25). If mLin-7 is localized by its interaction with mLin-2/CASK, what factors lead to the localization of mLin-2/CASK at the basolateral surface? In worm epithelia, lin-10 and lin-2 are essential for Let-23 localization and vulval formation, suggesting that Lin-10 interacts with Lin-2 and might be involved in Lin-2 localization. In mammalian cells, it is possible that X11 family members might also play a role in mLin-2/CASK localization to the basolateral surface. However, this hypothesis is challenged by several findings. First, the localization of X11 proteins does not always correlate with the localization of mLin-2/CASK. In neurons, we have identified X11alpha in a perinuclear region, most likely a part of the Golgi network (1). Similarly, we find that X11gamma is located in a perinuclear region in mammalian epithelia. However, although mLin-2/CASK colocalizes with X11alpha in neurons (1), mLin-2/CASK localized to the lateral membrane in epithelia. Second, although mLin-2/CASK interacts with X11alpha in the CNS, we could find no interaction between mLin-2/CASK with X11gamma in renal epithelia. It is possible that there is another form of X11 in epithelia that we have not yet detected that could control mLin-2/CASK targeting in cells. Another possibility is that that there are membrane components at the basolateral surface, distinct from X11, that bring mLin-2/CASK to the basolateral surface in mammalian epithelia. We also find that neither the NH2-terminal nor the COOH-terminal half of mLin-2/CASK is sufficient to correctly localize mLin-2/CASK. Thus the localization process for mLin-2/CASK may be complex and involve multiple interactions. The identification of the membrane components that localize mLin-2/CASK will yield important insights into the targeting of peripheral membrane proteins.


    ACKNOWLEDGEMENTS

We are grateful to Paul Welling for very helpful discussions, Mark Knepper for providing antibodies for use in the colocalization studies, Joseph Handler and H. Moo Kwon for BGT-1 cDNA, Juanita Merchant for the use of her fluorescent microscope, and Thomas Komorowski, manager of the University of Michigan Diabetes Research Center Morphology and Image Analysis Core, for assistance with confocal microscopy. We thank Jie Liu for extremely valuable technical assistance in carrying out immunolocalizations in the kidney.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-32839 (J. B. Wade), 5-T32-HD-07505 (S. W. Straight), and GM-08353 (D. Karnak). B. Margolis is an investigator of the Howard Hughes Medical Institute.

Present address: of J.-P. Borg: U119 INSERM, 27 Boulevard Lei Roure, 13009 Marseille, France.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. Margolis, Howard Hughes Medical Institute, Univ. of Michigan Medical Center, 4570 MSRB II, Box 0650, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0650 (E-mail: bmargoli{at}umich.edu).

Received 26 May 1999; accepted in final form 21 October 1999.


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