Title: Phosphorylation and Ankyrin-G Binding of the C-terminal Domain Regulate Targeting and Function of the Ammonium Transporter RhBG
Abstract: RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH3 transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the 419FLD421 sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH3 transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH3 transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain. RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH3 transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the 419FLD421 sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH3 transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH3 transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain. The protein homologues Rh, RhAG, RhBG, and RhCG are the four members of the human Rh 2The abbreviations used are: RhRhesusAmt/Mepammonia transporters/methylammonium-ammonium permeaseRhBG-Ctercytoplasmic C terminus of RhBGkAE1kidney type 1 anion exchangerPDZPSD-95 Discslarge ZO-1ZO-1zonula occludens-1HEKhuman embryonic kidneyMDCKMadin-Darby canine kidneymIMCDmurine inner medullary collecting ductGSTglutathione S-transferaseBCECF-AM2′7′-bis-(2-carboxyethyl)-5(6)-carboxyfluoresceinacetoxymethyl esterMFImean fluorescence intensityPBSphosphate-buffered saline. (Rhesus) family. They share a common predicted secondary structure with twelve transmembrane domains and both N and C termini located in the cytoplasm, a structure reminiscent of many membrane transporters (1Huang C.H. Liu P.Z. Blood Cells Mol. Dis. 2001; 27: 90-101Crossref PubMed Scopus (74) Google Scholar). Rh and RhAG are erythroid-specific membrane proteins and represent the “core” of the Rh membrane complex (2Avent N.D. 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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 these findings, crystallographic structure determination and transport experiments demonstrated that Escherichia coli AmtB is a channel that conducts uncharged NH3 (19Khademi S. O'Connell 3rd, J. Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (554) Google Scholar, 20Zheng L. Kostrewa D. Berneche S. Winkler F.K. Li X.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar). Based on the three-dimensional structure of AmtB, homology modeling emphasizing critical residues involved in the NH3 channel of the Rh protein family members has been proposed (21Callebaut I. Dulin F. Bertrand O. Ripoche P. Mouro I. Colin Y. Mornon J.P. Cartron J.P. Transfus. Clin. Biol. 2006; 13: 70-84Crossref PubMed Scopus (61) Google Scholar, 22Conroy M.J. 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Biosci. 2005; 10: 2832-2840Crossref PubMed Scopus (25) Google Scholar). We previously demonstrated that the Rh complex constitutes a major interaction site between the phospholipid bilayer and the erythrocyte membrane skeleton through direct binding of the cytoplasmic C-terminal tails of Rh and RhAG with ankyrin-R (29Nicolas 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 (113) Google Scholar). This finding led to the proposal of an erythroid model of the Rh/AE1 (type 1 anion exchanger) macrocomplex described by Bruce et al. (30Bruce 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 (285) Google Scholar), in which Rh complex proteins and AE1 could associate either directly or indirectly through their common interaction with ankyrin-R (29Nicolas 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 (113) Google Scholar). Moreover, we recently reported that the targeting and anchoring of human RhBG to the basolateral plasma membrane of epithelial kidney cells require a cis-tyrosine-based signal and an ankyrin-G-binding motif, respectively, both located in the cytoplasmic C-terminal tail (31Lopez C. Metral S. Eladari D. Drevensek S. Gane P. Chambrey R. Bennett V. Cartron J.P. Le Van Kim C. Colin Y. J. Biol. Chem. 2005; 280: 8221-8228Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) (Fig. 1). Although the structure of the cytosolic C termini of AmtB and Rh glycoproteins has not been resolved, this domain did not appear to be a central constituent of the gas channel. However, several mutational studies on both fungal (9Marini A.M. Urrestarazu A. Beauwens R. Andre B. Trends Biochem. 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Moreover, the recent structure of an Rh homologue from N. europaea revealed the presence of a cytoplasmic C-terminal helix that could regulate channel opening by interaction with a protein partner (23Li X. Jayachandran S. Nguyen H.H. Chan M.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19279-19284Crossref PubMed Scopus (72) Google Scholar). Alternatively, the crystal structure of the archeal Amt-1 strongly suggested that the C terminus interacts physically with cytosolic loops of the protein (35Andrade S.L. Dickmanns A. Ficner R. Einsle O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14994-14999Crossref PubMed Scopus (185) Google Scholar), and recent reports on the plant AtAMT1;1 (36Loque D. Lalonde S. Looger L.L. von Wiren N. Frommer W.B. Nature. 2007; 446: 195-198Crossref PubMed Scopus (213) Google Scholar) and AtAMT1;2 (37Neuhauser B. Dynowski M. Mayer M. Ludewig U. 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We investigated the role of phosphorylation and anchorage to the plasma membrane skeleton on surface expression and transport activity of RhBG. We propose a model of regulation of RhBG targeting and function by the cytoplasmic C-terminal tail. Materials—Primers used in PCR and mutagenesis experiments were from MWG Biotech (Ebersberg, Germany). The QuikChange XL site-directed mutagenesis kit and Escherichia coli BL21 and TKB1 strains were provided by Stratagene (La Jolla, CA). The pGEX-5X-3 vector, the protein A-Sepharose CL4B beads, and the glutathione-Sepharose 4B beads were purchased from Amersham Biosciences. Complete protease inhibitor mixture was supplied by Roche Applied Science. Purified Src and Syk kinases were provided by Cell Signaling Technology (Danvers, MA), and sodium orthovanadate was purchased from Calbiochem (Darmstadt, Germany). Wild-type and Mutant RhBG and RhBG-Cter Expression Vectors—The mutated human RhBG cDNAs S422A, S426A, T456A, S422D, S426D, Y429D, T456D, Y429E, Y429F, and 454Stop were obtained by in vitro mutagenesis from the pcDNA3-RhBG vector previously described (31Lopez C. Metral S. Eladari D. Drevensek S. Gane P. Chambrey R. Bennett V. Cartron J.P. Le Van Kim C. Colin Y. J. Biol. Chem. 2005; 280: 8221-8228Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), according to the supplier's instructions (Stratagene). The PCR-amplified cDNA fragment encoding the C-terminal tail of RhBG (RhBG-Cter, residues 416–458, starting from the ATG codon, Fig. 1) was inserted between the EcoRI and XhoI sites of the pGEX-5X-3 vector, in-frame with the DNA coding for the GST protein. The mutant form of RhBG-Cter Y429A was derived from pGEX-5X-3-RhBG-Cter by in vitro mutagenesis. All the inserts were sequenced using an ABI-PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). The pCEP4-RhBG vector, containing the full-length cDNA for RhBG and the hygromycin resistance gene as selection marker, was described previously (18Zidi-Yahiaoui N. Mouro-Chanteloup I. D'Ambrosio A.M. Lopez C. Gane P. Le van Kim C. Cartron J.P. Colin Y. Ripoche P. Biochem. J. 2005; 391: 33-40Crossref PubMed Scopus (73) Google Scholar). Antibodies—Rabbit polyclonal antiserum raised against human RhBG-Cter has been described previously (6Quentin 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 (133) Google Scholar). This antibody was affinity-purified using the SulfoLink kit from Pierce (Rockford, IL). A mouse polyclonal antibody was raised against the extracellular loops of RhBG by immunization with recombinant mIMCD-3 cells expressing human RhBG at their surface. The immunization schedule was as follows (Kernov, St. Etienne-en-Cogles, France): BALB/C SSC mice were immunized by three intraperitoneal injections of 2 × 106 cells, the first injection with Freund's complete adjuvant, the following injections with Freund's incomplete adjuvant. Ascites fluids were screened and checked for the specificity of the antibody by enzyme-linked immunosorbent assay and flow cytometry on both HEK293-RhBG transfectants and parental HEK293 cells; ascites A-08 was selected for further studies. The specificity of this mouse polyclonal antibody for RhBG protein was further demonstrated by flow cytometry (Table 1), immunofluorescence (Fig. 2B), and immunoprecipitation studies (below under “Experimental Procedures” and Fig. 7). Peroxidase-conjugated anti-rabbit and anti-mouse IgG were provided by P.A.R.I.S. (Compiègne, France). Mouse anti-human ZO-1 was purchased from Zymed Laboratories Inc. (San Francisco, CA). The monoclonal anti-phosphotyrosine antibodies P-Tyr-100 and P-Tyr-102 were provided by Cell Signaling Technology and 4G10 by Upstate (Charlottesville, VA). Alexa Fluor anti-rabbit and anti-mouse IgG were from Invitrogen. Erk1+Erk2 rabbit polyclonal antibody was provided by Abcam (Cambridge, UK).TABLE 1Cell surface expression and NH3 transport activity of C terminus mutants of RhBGCell clonesExpressionaRelative values to MFI for RhBGAlkalinization rate constantsTransport efficiencybRelative values of alkalinization rate constants corrected for membrane expression of RhBG and after subtraction of the passive diffusion constant (WT)%k(s–1)%WTcParental HEK293 cells00.22 ± 0.068 (14)dn values are in parentheses0RhBG1001.38 ± 0.215 (14)100EmptyeHEK293 cells transfected with an empty pcDNA3 vector00.24 ± 0.025 (3)2F419A/L420A/D421A1020.31 ± 0.087 (3)7S422A1021.05 ± 0.081 (4)70S422D700.84 ± 0.078 (4)76S426A1051.03 ± 0.072 (4)66S426D680.84 ± 0.065 (4)78Y429A1131.31 ± 0.278 (3)83Y429F981.32 ± 0.110 (4)97Y429D1150.28 ± 0.034 (3)5Y429E650.25 ± 0.028 (3)3454Stop710.88 ± 0.059 (3)79T456A1081.17 ± 0.221 (4)76T456D1041.19 ± 0.255 (4)81a Relative values to MFI for RhBGb Relative values of alkalinization rate constants corrected for membrane expression of RhBG and after subtraction of the passive diffusion constant (WT)c Parental HEK293 cellsd n values are in parenthesese HEK293 cells transfected with an empty pcDNA3 vector Open table in a new tab FIGURE 7Delivery and turnover of RhBG, Y429F, Y429E, and F419A/L420A/D421A at the surface of HEK293 cells. HEK293 cells expressing RhBG mutants of Tyr429 or ankyrin-G binding site were pulse-chase-labeled ([35S]methionine/[35S]cysteine), and total membrane proteins were biotinylated and immunoprecipitated. A, newly delivered RhBG proteins at the cell surface were monitored by autoradiography of biotinylated proteins. Total amounts of RhBG proteins expressed at the plasma membrane at each time of chase were determined by Western blot using anti-RhBG A-08. B, the curves show the ratio of radiolabeled biotinylated RhBG/total biotinylated RhBG at the membrane reflecting the turnover of newly delivered native or mutant RhBG at the cell surface as a function of time. The curves represent mean values from three experiments. ♦, RhBG; ▪ Y429F; ⋄, Y429E; ▵, F419A/L420A/D421A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell Culture, Transfection, Flow Cytometry, and Confocal Microscopy—Madin-Darby canine kidney (MDCK) cells, human embryonic kidney (HEK) 293 cells, and murine inner medullary collecting duct (mIMCD-3) cells were supplied by the American Type Culture Collection (Manassas, VA). MDCK cells were grown in Dulbecco's modified Eagle's medium/Glutamax I (Invitrogen), HEK293 and mIMCD-3 cells were grown in Iscove's modified Dulbecco's medium (Invitrogen), and all media were supplemented with 10% fetal calf serum (Dutscher, Brumath, France). Stable MDCK, HEK293, and mIMCD-3 cells expressing native or 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 (MDCK) or 0.8 mg/ml (HEK293) neomycin (Geneticin, Invitrogen), or 0.4 mg/ml hygromycin (Invitrogen) for mIMCD-3 cells. RhBG-positive cells were detected by flow-cytometry using the rabbit anti-RhBG-Cter polyclonal antibody after permeabilization of the cells, as described previously (31Lopez C. Metral S. Eladari D. Drevensek S. Gane P. Chambrey R. Bennett V. Cartron J.P. Le Van Kim C. Colin Y. J. Biol. Chem. 2005; 280: 8221-8228Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), or using the murine anti-RhBG ascites A-08 (1:200) without prior permeabilization and then cloned by limiting dilutions. At least three clones of each transfectant were grown and subsequently analyzed. Subconfluent (HEK293) or confluent (MDCK) monolayers of transfectants were cultured on poly-l-lysine coverslips or polycarbonate membranes, respectively, immunostained with the appropriate primary and Alexa Fluor secondary antibodies, and examined by confocal microscopy as described previously (31Lopez C. Metral S. Eladari D. Drevensek S. Gane P. Chambrey R. Bennett V. Cartron J.P. Le Van Kim C. Colin Y. J. Biol. Chem. 2005; 280: 8221-8228Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Stopped-flow Analysis—The ammonium transport function of wild-type RhBG and mutants was determined by stopped-flow spectrofluorometry analysis. Intracellular pH variation, using BCECF-AM (Sigma-Aldrich) as a pH-sensitive fluorescent probe, was measured in parental and transfected HEK293 cells exposed to inwardly directed 20 mmol of ammonium (NH+4) gradients at pH 7.2, as extensively described previously (18Zidi-Yahiaoui N. Mouro-Chanteloup I. D'Ambrosio A.M. Lopez C. Gane P. Le van Kim C. Cartron J.P. Colin Y. Ripoche P. Biochem. J. 2005; 391: 33-40Crossref PubMed Scopus (73) Google Scholar, 44Zidi-Yahiaoui N. Ripoche P. Le Van Kim C. Gane P. D'Ambrosio A.M. Cartron J.P. Colin Y. Mouro-Chanteloup I. Transfus. Clin. Biol. 2006; 13: 128-131Crossref PubMed Scopus (7) Google Scholar). When indicated, pervanadate was added at 0.1 mm to culture medium 30 min before cell trypsinization and during each step before analysis with the stopped-flow instrument (SFM3, Bio-Logic, Grenoble, France). Electrophoresis and Western Blot Analysis—SDS-PAGE was performed using 12.5% polyacrylamide gels according to Laemmli (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). Western blots were performed on nitrocellulose membranes, which then were stained with Ponceau Red (0.1%) and/or incubated with relevant primary antibodies followed with the appropriate peroxidase-conjugated secondary antibody (1:1000). Immunoblots were visualized using the ECL Plus Western blotting Detection System (Amersham Biosciences). In Vitro Phosphorylation Assays—The GST fusion proteins expressed in E. coli BL21 and TKB1 were purified by elution from glutathione-Sepharose 4B beads (150 mm NaCl, 50 mm Tris-HCl, pH 8, 20 mm glutathione) and quantified by absorption at 280 nm. For in vitro phosphorylation with purified kinases, 15 μg of GST-RhBG-Cter fusion protein produced in BL21 cells and bound to glutathione-Sepharose 4B beads were mixed with 100 ng of Src or Syk kinase in the reaction buffer: 60 mm HEPES, pH 7.5, 5 mm MgCl2, 5 mm MnCl2, 1 mm DTT, 3 μm sodium orthovanadate, and incubated with 10 μCi of [γ-32P]ATP (200 μm) for 30 min at 37 °C. Beads were washed six times with phosphate-buffered saline (PBS) containing 0.05% Triton X-100 and then resuspended and boiled for 5 min in 1× Laemmli buffer. Samples were subjected to 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Radioactive bands corresponding to GST-RhBG-Cter were visualized using a Bio Imaging Analyser BAS-1800 II (Fujifilm-Raytest, Courbevoie, France). Protein Extraction From HEK293 or MDCK Cells and Immunoprecipitation—Wild-type or transfected cells were lysed for 1 h at 4 °C in lysis buffer (150 mm NaCl, 20 mm Tris-HCl, pH 8, 5 mm EDTA) containing complete protease inhibitor mixture and usually 1% Triton X-100, or variable amounts when indicated. Lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Aliquots of lysates were mixed with 5× loading buffer (1.25 m sucrose, 20% SDS, 250 mm Tris-HCl, pH 6.8, 25% β-mercaptoethanol, 1% bromphenol blue) before electrophoresis. For RhBG immunoprecipitation, lysates were submitted to preclearing by incubation with protein A-Sepharose CL4B beads supplemented with 2% goat serum for 2 h at 4 °C and centrifuged at 1,000 × g for 5 min at 4 °C. Supernatants were incubated with 1 μg of purified rabbit anti-RhBG-Cter and protein A-Sepharose CL4B beads for 2 h at 4 °C. Beads were then washed three times with lysis buffer, and immunocomplexes were eluted in Laemmli buffer for 1 h at room temperature and mixed with loading buffer. Samples were electrophoresed in 12.5% SDS-PAGE gels and immunoblotted using murine ascites A-08 (1:5,000), which is directed against the extracellular loops of RhBG. The recognition of RhBG protein, immunoprecipitated by the anti-RhBG-Cter, actually demonstrated the specificity of ascites A-08 for RhBG. Membrane Targeting Assays of RhBG Protein—Delivery of newly synthesized RhBG proteins to the membrane and their turnover were determined by performing a pulse-labeled biotin targeting assay. HEK293 cells expressing native or mutated RhBG protein were grown to confluency on 60-mm Petri dishes. Cells were washed twice with Dulbecco's modified Eagle's medium minus methionine and cysteine and then incubated for 30 min in the same medium supplemented with 10% fetal calf serum. Newly synthesized proteins were labeled by adding 150 μCi of [35S]methionine/[35S]cysteine (Invitrogen) in the culture medium for 10 min at 37 °C. Cells were washed and incubated in nonradioactive medium for 0.5, 1.5, 3.5, 6, 10, 13, and 17 h at 37 °C. After each time, cells were washed twice with cold PBS and incubated with 0.5 mg/ml nonpenetrating Sulfo-NHS-LC-biotin (Pierce) diluted in biotinylation buffer (10 mm Hepes, 150 mm NaCl, 0.2 mm CaCl2, 0.2 mm MgCl2, pH 7.5) for 30 min at 4 °C. After removal of biotin, cells were incubated with 10 mm glycine for 10 min at 4 °C and washed twice with cold PBS. Cell lysis and immunoprecipitation of RhBG proteins were performed as described above. The immunocomplexes were washed twice with lysis buffer, and one-fourth (200 μl) was eluted in Laemmli buffer for 1 h at room temperature (total RhBG proteins). The remaining three-fourths