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• W2081009642 abstract "RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells. RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells. The Rhesus (Rh) 1The abbreviations used are: Rh, Rhesus; Mep/Amt, methylammonium-ammonium permease/ammonia transporters; AE1, type 1 anion exchanger; RhBG-Cter, cytoplasmic C terminus of RhBG; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; WT, wild type; ZO, zonula occludens.1The abbreviations used are: Rh, Rhesus; Mep/Amt, methylammonium-ammonium permease/ammonia transporters; AE1, type 1 anion exchanger; RhBG-Cter, cytoplasmic C terminus of RhBG; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; WT, wild type; ZO, zonula occludens. family is composed of four protein homologues, Rh, RhAG, RhBG, and RhCG, all of which are predicted to be polytopic transmembrane proteins with 12 membrane spanning domains and intracytoplasmic N and C termini (1Huang C.H. Liu P.Z. Blood Cells Mol. Dis. 2001; 27: 90-101Crossref PubMed Scopus (73) Google Scholar). Rh and RhAG are expressed on red cells only, where they define the core of the Rh membrane complex, which also includes the unrelated accessory chains CD47, LW/ICAM-4, and GPB (2Cartron J.P. Bailliere's Best Pract. Res. Clin. Haematol. 1999; 12: 655-689Crossref PubMed Scopus (104) Google Scholar, 3Avent N.D. Reid M.E. Blood. 2000; 95: 375-387Crossref PubMed Google Scholar). Conversely, RhBG and RhCG are not expressed in erythroid tissues but are both expressed in kidney and differentially in other organs as follows: testis, brain, pancreas, and prostate for RhCG and liver, skin, and ovary for RhBG (4Liu Z. Chen Y. Mo R. Hui C. Cheng J.F. Mohandas N. Huang C.H. J. Biol. Chem. 2000; 275: 25641-25651Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 5Liu Z. Peng J. Mo R. Hui C. Huang C.H. J. Biol. Chem. 2001; 276: 1424-1433Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Primary structure homology (30–35%) with the methylammonium-ammonium permease/ammonia transporters (Mep/Amt) from the lower organisms and plants has raised the possibility that Rh, RhAG, RhBG, and RhCG should represent the mammalian members of an Amt/Mep/Rh superfamily of ammonium transporters (1Huang C.H. Liu P.Z. Blood Cells Mol. Dis. 2001; 27: 90-101Crossref PubMed Scopus (73) Google Scholar, 6Marini A.M. Urrestarazu A. Beauwens R. Andre B. Trends Biochem. Sci. 1997; 22: 460-461Abstract Full Text PDF PubMed Scopus (214) Google Scholar, 7Weiner I.D. Curr. Opin. Nephrol. Hypertens. 2004; 13: 533-540Crossref PubMed Scopus (51) Google Scholar). Accordingly, ammonium ((NH4+) and/or NH3) transport capacity of RhAG, RhBG, and RhCG has been demonstrated in different heterologous expression systems (8Marini A.M. Matassi G. Raynal V. Andre B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (294) Google Scholar, 9Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 10Bakouh N. Benjelloun F. Hulin P. Brouillard F. Edelman A. Cherif-Zahar B. Planelles G. J. Biol. Chem. 2004; 279: 15975-15983Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Ludewig U. J. Physiol. (Lond.). 2004; 559: 751-759Crossref Scopus (106) Google Scholar). Moreover, recent ammonium transport analysis performed in red blood cells from human and mouse genetic variants with Rh and/or RhAG deficiencies indicated that RhAG facilitates NH3 movement across the red blood cell membrane and thus represents a potential example of gas transporter in mammalian cells (12Ripoche P. Bertrand O. Gane P. Birkenmeier C. Colin Y. Cartron J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17222-17227Crossref PubMed Scopus (141) Google Scholar). Supporting this finding, three-dimensional structure elucidation and transport experiments in an acellular model demonstrated that bacterial AmtB is a channel that conducts uncharged NH3 (13Khademi S. O'Connell III, J. Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (542) Google Scholar). Rh proteins also represent, at least in red blood cells, important structural components of the cell membrane, as first evidenced by the morphological abnormalities (stomato-spherocytosis) of Rhnull red cells that lack Rh and/or RhAG (2Cartron J.P. Bailliere's Best Pract. Res. Clin. Haematol. 1999; 12: 655-689Crossref PubMed Scopus (104) Google Scholar). It has been demonstrated recently that the Rh complex constitutes, as the previously described AE1-protein 4.2-ankyrin and GPC-protein 4.1-p55 complexes (14Bhattacharyya R. Das A.K. Moitra P.K. Pal B. Mandal I. Basu J. Biochem. J. 1999; 340: 505-512Crossref PubMed Scopus (17) Google Scholar, 15Nunomura W. Takakuwa Y. Parra M. Conboy J.G. Mohandas N. J. Biol. Chem. 2000; 275: 6360-6367Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), a major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton, and that the disruption of this interaction results in part in the morphological abnormalities of Rhnull red cells (16Gane P. Le Van Kim C. Bony V. El Nemer W. Mouro I. Nicolas V. Colin Y. Cartron J.P. Br. J. Haematol. 2001; 113: 680-688Crossref PubMed Scopus (32) Google Scholar, 17Mouro-Chanteloup I. Delaunay J. Gane P. Nicolas V. Johansen M. Brown E.J. Peters L.L. Van Kim C.L. Cartron J.P. Colin Y. Blood. 2003; 101: 338-344Crossref PubMed Scopus (99) Google Scholar, 18Nicolas V. Le Van Kim C. Gane P. Birkenmeier C. Cartron J.P. Colin Y. Mouro-Chanteloup I. J. Biol. Chem. 2003; 278: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The Rh complex interacts with the erythrocyte skeleton through direct binding of the cytoplasmic C-terminal tails of Rh and RhAG with the second repeat subdomain (D2) of the membrane binding domain of ankyrin-R (18Nicolas V. Le Van Kim C. Gane P. Birkenmeier C. Cartron J.P. Colin Y. Mouro-Chanteloup I. J. Biol. Chem. 2003; 278: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Based on these results and on the observation that expression of Rh proteins and AE1 (type 1 anion exchanger) is collectively decreased in ankyrin-R-deficient erythrocytes, we proposed a model of the Rh-AE1 macrocomplex described by Bruce et al. (19Bruce L.J. Beckmann R. Ribeiro M.L. Peters L.L. Chasis J.A. Delaunay J. Mohandas N. Anstee D.J. Tanner M.J. Blood. 2003; 101: 4180-4188Crossref PubMed Scopus (281) Google Scholar), in which Rh complex proteins and AE1 could associate either directly or indirectly through their common interaction with ankyrin-R (18Nicolas V. Le Van Kim C. Gane P. Birkenmeier C. Cartron J.P. Colin Y. Mouro-Chanteloup I. J. Biol. Chem. 2003; 278: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Ankyrins constitute a family of membrane adaptor proteins that link diverse membrane-associated proteins (ions channels and cell adhesion molecules) to the spectrin-based membrane skeleton. In vertebrates, three different genes Ank1, Ank2, and Ank3 encode ankyrin-R, ankyrin-B, and ankyrin-G, respectively. Ankyrin-R expression is restricted to erythrocytes, neurons, and striated muscle; ankyrin-B is broadly expressed and represents the major form in the nervous system, and ankyrin-G is the most widely distributed species and is predominant in epithelial cells (20Bennett V. Baines A.J. Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (774) Google Scholar). These last two forms are not found in red cells. In nonerythroid cells, the structural role of RhBG and RhCG has not been evaluated yet. However, their interactions with other membrane proteins and especially with cytoskeletal proteins are expected to be important for their correct location in functional membrane microdomains. We and others have shown that the epithelial expression of RhBG and RhCG in the kidney is restricted to the cells involved in ammonium secretion (connecting tubule and collecting duct) and that both proteins are expressed in the same cell types but with opposite polarity; RhBG is localized at the basolateral domain of the plasma membrane and RhCG at the apical domain (21Eladari D. Cheval L. Quentin F. Bertrand O. Mouro I. Cherif-Zahar B. Cartron J.P. Paillard M. Doucet A. Chambrey R. J. Am. Soc. Nephrol. 2002; 13: 1999-2008Crossref PubMed Scopus (116) Google Scholar, 22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar, 23Verlander J.W. Miller R.T. Frank A.E. Royaux I.E. Kim Y.H. Weiner I.D. Am. J. Physiol. 2003; 284: F323-F337Crossref PubMed Scopus (154) Google Scholar). Which signal(s) within the molecules determines their specific polarity remains unknown. However, considering the erythroid Rh-ankyrin R complex discussed above and previous observations (24Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar, 25Piepenhagen P.A. Peters L.L. Lux S.E. Nelson W.J. Am. J. Physiol. 1995; 269: C1417-C1432Crossref PubMed Google Scholar, 26Doctor R.B. Chen J. Peters L.L. Lux S.E. Mandel L.J. Am. J. Physiol. 1998; 274: F129-F138Crossref PubMed Google Scholar) indicating that ankyrin-G is located at the basolateral side of kidney tubular epithelial cells, we hypothesized that the basolateral epithelial homologue RhBG could interact with the spectrin-based skeleton of epithelial cells through homologues of ankyrin-R, i.e. ankyrin-B and/or ankyrin-G (18Nicolas V. Le Van Kim C. Gane P. Birkenmeier C. Cartron J.P. Colin Y. Mouro-Chanteloup I. J. Biol. Chem. 2003; 278: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In this study, we analyzed the sorting and tethering of RhBG to the basolateral membrane of renal epithelial cells. We identify both cis determinants required for the targeting of RhBG and the trans factor(s) necessary for its anchorage into the membrane. Materials—Primers used in PCR and mutagenesis experiments were from MWG Biotec (Ebersberg, Germany). The QuikChange XL site-directed mutagenesis kit was provided by Stratagene (La Jolla, CA). The pcDNA3 vector was purchased from Invitrogen. The pGBKT7 and pGADT7 vectors were from the Matchmaker Gal4 Two-hybrid System 3 (Clontech). Rabbit polyclonal antiserum raised against the cytoplasmic C-terminal tail of human RhBG (RhBG-Cter) and affinity-purified rabbit polyclonal antibody raised against the C-terminal domain of human ankyrin-B were described previously (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar, 27Scotland P. Zhou D. Benveniste H. Bennett V. J. Cell Biol. 1998; 143: 1305-1315Crossref PubMed Scopus (145) Google Scholar). Mouse anti-human ankyrin-G and ZO-1 monoclonal antibodies (mAbs) were purchased from Zymed Laboratories Inc.. Mouse mAb to rat AE1 (28Alper S.L. Stuart-Tilley A.K. Biemesderfer D. Shmukler B.E. Brown D. Am. J. Physiol. 1997; 273: F601-F614PubMed Google Scholar) was kindly provided by Dr. Daniel Biemesderfer (Yale University, New Haven, CT). Construction of RhBG Mammalian Expression Vectors and Mutagenesis—Full-length cDNA encoding RhBG was amplified by PCR from human kidney Marathon-Ready cDNA (Clontech) by using published information (5Liu Z. Peng J. Mo R. Hui C. Huang C.H. J. Biol. Chem. 2001; 276: 1424-1433Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), sequenced on both strands using an ABI PRISM 310 Genetic Analyser (Applied Biosystem, Warrington, UK), and subcloned in a pcDNA3 expression vector. The mutated RhBG cDNAs Y429A/E430A/D431A/Q432A, Y429A, E430A, D431A, Q432A, F419A/L420A/D421A, and Y429A+F419A/L420A/D421A were constructed by in vitro mutagenesis from pcDNA3-RhBG double strand recombinant DNA according to the supplier's instructions (Stratagene). Cell Culture, Transfection, and Flow Cytometric Analysis—Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium/Glutamax I (Invitrogen) supplemented with 10% fetal calf serum. Stable MDCK transfectants expressing wild-type and mutated RhBG proteins were obtained after transfection with the relevant expression vectors using Lipofectin reagent (Invitrogen) and selection in culture medium supplemented with 0.6 mg/ml neomycin (Geneticin, Invitrogen). RhBG-positive cells were detected by flow cytometry using a FACSCalibur™ flow cytometer (BD Biosciences), after permeabilization/fixation by 50% ethanol in phosphate-buffered saline (PBS) and staining with rabbit polyclonal anti-RhBG-Cter (1:500), as primary antibody, and donkey anti-rabbit phycoerythrin-conjugated F(ab′)2 fragments (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Stable clones expressing RhBG were obtained after limiting dilutions, and at least two clones of each transfectant were grown and subsequently analyzed. Immunofluorescence and Confocal Microscopy on RhBG-expressing MDCK Cells—Subconfluent or confluent monolayers of MDCK transfectants were cultured on 12-mm diameter poly-l-lysine coverslips (BD Biosciences) for 3 days or 12-mm diameter Transwell polycarbonate membranes (pore size, 0.4 μm) (Corning Costar, Cambridge, MA) for 7 days, respectively, prior to immunostaining. Cells were fixed in 4% paraformaldehyde for 20 min and washed in PBS. Free aldehyde groups were blocked by 50 mm NH4Cl for 10 min, and cells were then washed in PBS and permeabilized in 1% SDS for 15 min. After two washings in PBS, cells were incubated with rabbit polyclonal anti-RhBG-Cter antibody (1:500) and, for some experiments, with murine anti-ankyrin-G (2.5 μg/ml) or anti-ZO-1 (4 μg/ml) mAbs diluted in background reducing buffer (Dako Corp., Copenhagen, Denmark) for 1 h at room temperature. Cells were washed three times in PBS containing 0.5% bovine serum albumin and incubated for 1 h at room temperature with Alexa Fluor 488 goat anti-rabbit IgG and, for ankyrin-G or ZO-1 staining, Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes, Leiden, The Netherlands) diluted 1:200 in PBS/bovine serum albumin. Samples were examined by wide field (poly-l-lysine coverslips) or confocal (Transwell membranes) microscopy using a Nikon Eclipse TE300 inverted microscope equipped with a 60× oil immersion objective NA 1.4 and a D-Eclipse C1 confocal system. Immunohistochemistry on Kidney Tissue—Rat kidneys were fixed in vivo by perfusion of 4% paraformaldehyde in Dulbecco's modified Eagle's/F-12 medium (Invitrogen). Coronal kidney sections containing all kidney zones were then post-fixed for 4–6 h at 4 °C in 4% paraformaldehyde and then embedded in paraffin. Subsequently, 4-μm sections of the paraffin block were deparaffinized in toluene and rehydrated through 99% ethanol. Rehydration was completed in Tris-buffered saline, pH 7.6 (TBS). Slides were then placed in a plastic tank filled with 1 mm EDTA, pH 8.0, and heated for 40 min at 96–98 °C in a water bath. This step unmasked antigen and allowed immunostaining on paraformaldehyde-fixed paraffin sections, as determined in preliminary experiments (not shown). To reduce nonspecific binding, sections were rinsed in TBS for 10 min and preincubated for 20 min with background reducing buffer (Dako Corp.). Rat kidney sections were double-labeled with either the rabbit polyclonal anti-human RhBG-Cter antibody, which also recognizes the rat isoform (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar) and the mouse mAb against human ankyrin-G, or with the rabbit polyclonal antibody against human ankyrin-B and the mouse mAb against rat AE1. The double labeling procedure was as follows: anti-RhBG or anti-ankyrin-B antibodies, diluted in background reducing buffer (1:200 or 1:1000, respectively), were applied for 2 h at room temperature. After three washes, sections were incubated with a 1:400 dilution (in background reducing buffer) of goat anti-rabbit IgG coupled to Cy2 (Amersham Biosciences) in TBS, 30 min at room temperature, followed by three TBS washes. The second round of labeling was then performed. Sections were incubated for 2 h at room temperature with either the mouse anti-ankyrin-G mAb or the mouse anti-AE1 mAb at a dilution of 1:200 or 1:20, respectively (in background reducing buffer), followed by goat biotinylated anti-mouse IgG (diluted 1:400) and Cy5-conjugated streptavidin (diluted 1:1000) (Amersham Biosciences) for 30 min each. Sections were mounted and then examined by confocal microscopy. Cy2 was excited at 488 nm and detected at 498–550 nm, and then, on exactly the same field, Cy5 was excited at 647 nm and detected at 663–758 nm. Yeast Two-hybrid Studies—The D1, D2, D3, and D4 repeat subdomains of the membrane binding domain of ankyrin-G and ankyrin-B were amplified by PCR using plasmids bearing relevant cDNA sequences of Ank3 (gift from Dr. E. Kordeli, Institut Jacques Monod, Paris, France) and Ank2 genes, respectively, as templates. They were then inserted between the EcoRI and BamHI sites of pGADT7 vector in-frame with the GAL4 activation domain. A cDNA fragment encoding the cytoplasmic C-terminal tail of RhBG (RhBG-Cter) was amplified by PCR and inserted between the EcoRI and BamHI sites of pGBKT7 vector to generate a fusion gene with the DNA binding domain of GAL4. Mutant forms of RhBG-Cter were obtained by site-directed mutagenesis (see “Results”). The pGBKT7 and pGADT7 constructs were cotransformed into the AH109 yeast strain using the lithium acetate method (29Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2008) Google Scholar). After 4 days of growth at 30 °C, colonies were streaked onto plates lacking adenine, histidine, leucine, and tryptophan. Protein interactions were analyzed 3 days following plating and confirmed by β-galactosidase assays as recommended by the manufacturer (Matchmaker Gal4 Two-hybrid System 3, Clontech). Basolateral Expression of Recombinant RhBG in MDCK Cells—We showed previously by immunocytochemistry that recombinant RhBG was readily expressed at the plasma membrane of transfected HEK293 cells (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar). In order to assess the polarized expression of RhBG in a heterologous system, the canine kidney epithelial MDCK cell line was stably transfected with the RhBG-pcDNA3 expression vector. Cell clones strongly expressed recombinant RhBG at the plasma membrane, as demonstrated by flow cytometry (mean fluorescence intensity∼500) and wide field microscopy, using the polyclonal anti-Cter antibody (not shown). Confluent monolayers of clones expressing RhBG were filter-grown, allowed to polarize, and analyzed by confocal microscopy (Fig. 1B, WT panel). XY horizontal sections clearly showed a basolateral labeling of RhBG protein. XZ vertical sections confirmed that RhBG was exclusively localized at the basolateral membrane (Fig. 1B, green) below the tight junctions marked by ZO-1 staining (red), in accordance with the in vivo basolateral expression of the protein at the plasma membrane of renal epithelial cells (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar, 23Verlander J.W. Miller R.T. Frank A.E. Royaux I.E. Kim Y.H. Weiner I.D. Am. J. Physiol. 2003; 284: F323-F337Crossref PubMed Scopus (154) Google Scholar). Therefore, MDCK cells represent an appropriate ex vivo model for further analyzing the basolateral sorting of RhBG. Mapping of the Basolateral Sorting Signal in the Cytoplasmic C Terminus of RhBG—Most of the basolateral targeting cis determinants rely either on tyrosine- or on dileucine-dependent motifs and are located in the cytoplasmic tails of basolaterally expressed proteins (30Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1325) Google Scholar). RhBG protein is predicted to contain both cytoplasmic N (13 amino acids) and C termini (43 amino acids) (1Huang C.H. Liu P.Z. Blood Cells Mol. Dis. 2001; 27: 90-101Crossref PubMed Scopus (73) Google Scholar, 5Liu Z. Peng J. Mo R. Hui C. Huang C.H. J. Biol. Chem. 2001; 276: 1424-1433Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The short N terminus does not exhibit either motif, whereas the C terminus shows a potential tyrosine-based basolateral determinant, YEDQ, at positions 429–432 of the protein sequence (Fig. 1A). However, this motif, with a neutral polar amino acid (Gln) at position Y+3, deviates from the standard sequence YXXØ, where Ø is a bulky hydrophobic amino acid. To test the functionality of this potential determinant, we constructed a mutant in which the four residues were substituted into alanine (Fig. 1A, Y429A/E430A/D431A/Q432A). Confocal microscopy (Fig. 1B) revealed that the mutant RhBG protein was expressed in a nonpolarized manner on both apical and basolateral surfaces (green labeling surrounding and below ZO-1 red staining, respectively, in the XZ section) and also accumulated partly on cytoplasmic membranes (as seen by intracellular granulations in both horizontal and vertical sections). Thus, basolateral expression of RhBG indeed depends on the tyrosine-based motif localized in the cytoplasmic C terminus of the protein. In order to evaluate the relative importance of each amino acid, we then generated mutants containing only one alanine substitution for either residue of the targeting determinant (Fig. 1A, Y429A, E430A, D431A, and Q432A). The Y429A, E430A, and D431A mutants all exhibited nonpolarized expression patterns and a much weaker intracellular retention than the complete Y429A/E430A/D431A/Q432A mutant (Fig. 1B), indicating that the three residues are necessary and have additive effects on the targeting machinery. In contrast, the Q432A mutant displayed a polarized expression similar to the basolateral wild-type RhBG (Fig. 1B). Interaction of RhBG with Ankyrin-G and Ankyrin-B in Yeast Two-hybrid Analysis—Rh and RhAG have been shown to interact with ankyrin-R through their cytoplasmic tails (18Nicolas V. Le Van Kim C. Gane P. Birkenmeier C. Cartron J.P. Colin Y. Mouro-Chanteloup I. J. Biol. Chem. 2003; 278: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In order to investigate whether the nonerythroid homologue(s) of ankyrin-R could be partner(s) of RhBG, the C terminus of RhBG was fused in-frame to the GAL4 DNA binding domain of the yeast two-hybrid pGBKT7 vector, and the four repeat domains of ankyrin-G and ankyrin-B (D1, D2, D3, and D4) were individually fused in-frame with the GAL4 activation domain of the pGADT7 vector. The recombinant vectors were cotransformed in the AH109 yeast strain, and their ability to grow in high stringency selective medium lacking adenine, histidine, leucine, and tryptophan was analyzed (Fig. 2, A and B). These experiments, confirmed by β-galactosidase activity assays (not shown), indicated that the C-terminal cytoplasmic tail of RhBG (RhBG-Cter) specifically interacted with the D3 and D4 repeat domains of ankyrin-G and only the D3 repeat domain of ankyrin-B. No association between RhBG-Cter and the remaining D1 and D2 repeat domains of ankyrin-G or the D1, D2, and D4 repeat domains of ankyrin-B could be detected. RhBG Colocalizes with Ankyrin-G, but Not Ankyrin-B, in Rat Kidney Epithelial Cells—To test in vivo the relevance of the interactions detected in yeast two-hybrid assays between RhBG and ankyrin-G or ankyrin-B, we next investigated whether ankyrin-G or ankyrin-B exhibited the same tubular and plasma membrane location as RhBG by indirect immunofluorescence of paraformaldehyde-fixed paraffin rat kidney sections. Fig. 3 (A–I) shows the localization of ankyrin-G (red) with respect to RhBG protein (green). As we reported previously (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar), RhBG was exclusively expressed in the basolateral membrane of the connecting tubule (Fig. 3, B and C) and cortical collecting duct epithelial cells (not shown) and in the basolateral membrane of intercalated cells of medullary collecting ducts (Fig. 3, E, F, H, and I). When the rat kidney sections were stained with anti-ankyrin-G antibody, we found the same broad pattern of expression as described previously by others (24Peters L.L. John K.M. Lu F.M. Eicher E.M. Higgins A. Yialamas M. Turtzo L.C. Otsuka A.J. Lux S.E. J. Cell Biol. 1995; 130: 313-330Crossref PubMed Scopus (130) Google Scholar, 25Piepenhagen P.A. Peters L.L. Lux S.E. Nelson W.J. Am. J. Physiol. 1995; 269: C1417-C1432Crossref PubMed Google Scholar, 26Doctor R.B. Chen J. Peters L.L. Lux S.E. Mandel L.J. Am. J. Physiol. 1998; 274: F129-F138Crossref PubMed Google Scholar). Briefly, proximal convoluted tubules (Fig. 3, A and B), thick ascending limbs of Henle's loop (Fig. 3, D and E), and distal convoluted tubules (not shown) exhibited a strong and diffuse labeling of the basolateral domain of tubular epithelial cells, whereas more discrete basolateral labeling was observed in the connecting tubules (Fig. 3, A and B) and the collecting ducts (Fig. 3, D, E, G, and H). Finally, no ankyrin-G staining was detected in the glomeruli and the thin limbs (not shown). As illustrated in Fig. 3, B, E and H, simultaneous examination of ankyrin-G and RhBG stainings demonstrated that all RhBG-positive cells also expressed ankyrin-G. More importantly, within the outer medullary collecting duct and the inner medullary collecting duct, where we have previously shown that RhBG expression is restricted to intercalated cells (22Quentin F. Eladari D. Cheval L. Lopez C. Goossens D. Colin Y. Cartron J.P. Paillard M. Chambrey R. J. Am. Soc. Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (131) Google Scholar), we observed a perfect overlap of RhBG and ankyrin-G staining (Fig. 3, E and H), and ankyrin-G was absent or only weakly expressed in RhBG-negative principal (Fig. 3E) or inner medullary collecting duct (Fig. 3H) cells. We next determined whether ankyrin-B is also present in R" @default.
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