G-protein-coupled Receptor-mediated Traffic of Na,K-ATPase to the Plasma Membrane Requires the Binding of Adaptor Protein 1 to a Tyr-255-based Sequence in the α-Subunit

Abstract

Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase α1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase α1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase α1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase α1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA α1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors. Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase α1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase α1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase α1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase α1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA α1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors. The dopaminergic and renin-angiotensin systems act coordinately to regulate blood pressure, in part, by affecting sodium transport in renal proximal tubules (1Aperia A.C. Annu Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (230) Google Scholar, 2Leong P.K. Devillez A. Sandberg M.B. Yang L.E. Yip D.K. Klein J.B. McDonough A.A. Am. J. Physiol. 2006; 290: F854-F863Crossref PubMed Scopus (50) Google Scholar, 3Zeng C. Eisner G.M. Felder R.A. Jose P.A. Curr. Med. Chem. Cardiovasc. Hematol. Agents. 2005; 3: 69-77Crossref PubMed Scopus (30) Google Scholar). The apical Na/H-exchanger and basolateral Na,K-ATPase (NKA) 2The abbreviations used are: NKA, Na,K-ATPase; Ang II, angiotensin II; AP-1, adaptor protein 1; AT1R, type 1 angiotensin II receptor; D1R, type 1 dopamine receptor; IP, immunoprecipitation; FRET, fluorescence resonance energy transfer; FRETN, normalized FRET; FRETC, corrected FRET; WT, wild-type; PMA, phorbol 12-myristate 13-acetate. are responsible for ∼75% of the renal-filtered sodium reabsorption (4Jose P.A. Eisner G.M. Felder R.A. Pharmacol. Ther. 1998; 80: 149-182Crossref PubMed Scopus (204) Google Scholar, 5Moe O.W. J. Am. Soc. Nephrol. 1999; 10: 2412-2425Crossref PubMed Google Scholar). By maintaining a low intracellular sodium concentration, Na,K-ATPase provides the driving force for renal proximal tubule sodium reabsorption (6Féraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (430) Google Scholar). Both the Na/H-exchanger and NKA are regulated by the hormones dopamine and angiotensin II (Ang II). Dopamine activation of type 1 dopamine receptors (D1Rs) induces natriuresis and inhibits renal proximal tubule NKA activity (1Aperia A.C. Annu Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (230) Google Scholar, 6Féraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (430) Google Scholar, 7Chen C.J. Lokhandwala M.F. Pharmacol. Toxicol. 1992; 70: 11-17Crossref Scopus (11) Google Scholar, 8Hussain T. Lokhandwala M.F. Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (171) Google Scholar, 9Kansra V. Chen C. Lokhandwala M.F. Eur. J. Pharmacol. 1995; 289: 391-394Crossref PubMed Scopus (38) Google Scholar, 10Zhang A. Devarajan P. Dorfman A.L. Morrow J.S. J. Biol. Chem. 1998; 273: 18681-18684Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The antinatriuretic Ang II, through activation of type 1 angiotensin II receptors (AT1Rs), stimulates sodium reabsorption in renal proximal tubule cells (11Cheng H.F. Becker B.N. Harris R.C. J. Clin. Investig. 1996; 97: 2745-2752Crossref PubMed Scopus (79) Google Scholar, 12Inagami T. Kambayashi Y. Ichiki T. Tsuzuki S. Eguchi S. Yamakawa T. Clin. Exp. Pharmacol. Physiol. 1999; 26: 544-549Crossref PubMed Scopus (63) Google Scholar, 13Navar L.G. Harrison-Bernard L.M. Imig J.D. Wang C.T. Cervenka L. Mitchell K.D. J. Am. Soc. Nephrol. 1999; 10: S266-S272PubMed Google Scholar, 14Navar L.G. Lewis L. Hymel A. Braam B. Mitchell K.D. J. Am. Soc. Nephrol. 1994; 5: 1153-1158Crossref PubMed Google Scholar) and activates NKA (15Aperia A. Holtback U. Syren M.L. Svensson L.B. Fryckstedt J. Greengard P. FASEB J. 1994; 8: 436-439Crossref PubMed Scopus (239) Google Scholar, 16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We have demonstrated that NKA regulation by dopamine and Ang II is due to a reduced/increased number of NKA molecules at the plasma membrane and not to modulation of the intrinsic NKA activity (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). At the cellular level, Ang II-dependent activation of NKA is mediated by recruitment of NKA molecules from intracellular compartments to the plasma membrane via a clathrin-coated vesicle-dependent mechanism (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Pedemonte C.H. Efendiev R. Bertorello A.M. Semin. Nephrol. 2005; 25: 322-327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Protein kinase Cβ-dependent phosphorylation of the NKA α1-subunit constitutes the triggering signal for NKA plasma membrane recruitment. Phosphorylation of Ser-11 and Ser-18 in NKA α1 may induce a conformational change in the protein to facilitate the binding of adaptor protein 1 (AP-1). Although we are beginning to better understand the organization of signals initiated by stimulation of AT1Rs and the traffic of NKA molecules from intracellular compartments to the plasma membrane, the mechanism involved in cargo selection remains unclear. The present study was performed to identify the NKA α1 sequence that interacts with AP-1. Materials—Ouabain, angiotensin II, chelerythrine chloride, and mouse anti-AP-1 antibody were obtained from Sigma. Alexa Fluor 488-tagged anti-mouse and Cy3-tagged anti-rabbit antibodies were purchased from Molecular Probes (Eugene, OR). N-hydroxysulfosuccinimide-biotin was obtained from Pierce. ExactaCruz E antigen detection system was purchased from Santa Cruz Biotechnology. Anti-Na,K-ATPase α1 monoclonal mouse antibody was a generous gift of Dr. Robert W. Mercer (Washington University, St. Louis, MO). Anti-Na,K-ATPase α1 polyclonal rabbit antibody was a generous gift of Dr. Jack H. Kaplan (University of Illinois, Chicago, IL). Other reagents were of the highest quality available. Cell Culture and Transfection—OK cells stably expressing the wild-type rat Na,K-ATPase α1 or mutant forms of this protein were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum and antibiotics. To prevent the expression of the endogenous NKA α1, cells were maintained all the time in 3 μm ouabain medium (18Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (38) Google Scholar, 19Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (71) Google Scholar). Plasmid preparation and site-directed mutagenesis were performed as described previously (20Done S.C. Leibiger I.B. Efendiev R. Katz A.I. Leibiger B. Berggren P.O. Pedemonte C.H. Bertorello A.M. J. Biol. Chem. 2002; 277: 17108-17111Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Stable expression of Na,K-ATPase WT α1 and mutants was performed as described by Pedemonte et al. (18Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (38) Google Scholar, 19Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (71) Google Scholar). Cell Treatment—The optimal concentrations of the reagents and the extension of the treatments used in these experiments were as determined previously (21Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Feraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (84) Google Scholar, 22Efendiev R. Bertorello A.M. Zandomeni R. Cinelli A.R. Pedemonte C.H. J. Biol. Chem. 2002; 277: 11489-11496Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 23Efendiev R. Pedemonte C.H. J. Am. Soc. Nephrol. 2006; 17: 31-38Crossref PubMed Scopus (21) Google Scholar). In some experiments, before and during the treatment with hormones, the cells were incubated with 2 μm chelerythrine chloride for 30 min to inhibit protein kinase C activity. Determination of Rb+ Uptake—Measurements of NKA-mediated transport by Rb+ uptake were performed with attached cells as described previously (18Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (38) Google Scholar, 19Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (71) Google Scholar). Cells were transferred to serum-free Dulbecco's modified Eagle's medium containing 50 mm HEPES, pH 7.4, and either 3 μm or 5 mm ouabain (incubation medium). All treatments and determinations were performed at 23 °C. Then, a trace amount of [86Rb]RbCl was added to the cell medium. After 20 min, cells were washed three times with ice-cold saline and dissolved with SDS, and accumulated radioactivity was determined. NKA-mediated Rb+ uptake was calculated from the difference in tracer uptake between samples incubated in 3 μm or 5 mm ouabain. The ouabain-insensitive Rb+ uptake (measured in the presence of 5 mm ouabain) was 25-30% of the total Rb+ uptake measured. In some experiments, cells were with hormones before the Rb+ uptake The concentrations used and the time of treatment are described in the Determination of of NKA by experiments, the cells were with 1 μm or 1 Ang II for min at and the medium was to ice-cold mm pH 2 mm mm and After for 1 at the cells were in mm 2 mm 2 mm and 30 mm sodium pH containing a in in and The cell was at at for 5 The was and protein was determined. containing of protein were transferred to and and were The was incubated at for 1 with antibody and for 2 with protein three times with saline and with containing After the were washed times with containing and and with 50 mm pH 7.4, and in with and were performed as described previously (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, R. Bertorello A.M. Pressley T.A. Rousselot M. Feraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (84) Google Scholar, R. J. Leibiger B. Katz A.I. Berggren P.O. Pedemonte C.H. Leibiger I.B. Bertorello A.M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Determination of with NKA cells stably expressing either the WT or mutant of NKA α1 were with 1 Ang II for min at The cells were in ice-cold by the ExactaCruz as described by the Cell amount of protein for were with E Cruz for 30 min at C. mouse serum and antibody reagents are from Santa Cruz and and NKA α1 antibody were incubated with the ExactaCruz to were washed with saline and incubated with cell for 1 at °C. The were washed three times with and in After by the proteins were transferred to a of membrane, was by with The protein were by The membrane was and with α1 antibody to determine NKA as a of The of integral of protein by and NKA α1 antibodies in the membrane was calculated for Then, the membrane was and with antibody to determine the level of NKA α1 of Cells for attached to and to were incubated for min at with 1 Ang II in serum-free Dulbecco's modified Eagle's medium containing 50 mm HEPES, pH The cells were washed with saline containing with in for min, washed with incubated with for 20 min, washed with with serum and in for min, and washed with The cells were with serum and 20 for 1 Cells were incubated with mouse anti-AP-1 and rabbit α1 antibodies and washed with In the cells were incubated with Alexa Fluor 488-tagged anti-mouse and Cy3-tagged anti-rabbit antibodies for 1 Then, the were and containing of of the the fluorescence of fluorescence with a a a and and a for of and and all by To of at of the with a of μm were and was performed all the of was were through three the Alexa Fluor and Alexa Fluor was calculated a for the the Alexa Fluor B. B. J. 1998; Full Text Full Text PDF PubMed Scopus Google FRET, Alexa Fluor and to of cells with Alexa Fluor and through the FRET, Alexa Fluor and of and are the that were calculated for the of Alexa Fluor and through the of were performed in cells with of the either Fluor or In the with of normalized were calculated to the Fluor FRETC, Alexa Fluor and are the of FRETC, Alexa Fluor and Y. J. 2001; 81: Full Text Full Text PDF PubMed Scopus Google Scholar). were obtained the from are with low and was at three between the two was determined with the was with in the the at of cells were used in The for proteins as with proteins through binding to protein the and are and and or the used is are to and is a residue with a J.S. J. Cell Biol. 1999; PubMed Scopus Google Scholar). The sequence of may in the of the sequence of the NKA the presence of of these within the of α1 at and The of the of the by the in the α1 were to The the change by the and the of the residue containing the rat wild-type NKA α1 and forms were stably in a cell that is used as a of renal proximal tubule J. M. M. P. J. Cell Biol. 1997; PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar, Mol. Pharmacol. Google Scholar). Cells expressing the WT and mutant forms of NKA α1 were and maintained all the time in the presence of 3 μm ouabain medium to prevent the expression of the endogenous α1-subunit C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (71) Google Scholar, C.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). activity mediated by the NKA was determined in these The Rb+ uptake in WT α1 cells was and were determined in cells expressing the different NKA α1 that the NKA was at the level in the different cell Ang II-dependent stimulation of AT1Rs (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) the Rb+ uptake to protein in cells expressing the wild-type NKA α1 transport were in of the NKA α1 the stimulation was in cells expressing the NKA α1 mutant We have previously demonstrated that a receptor and the phorbol PMA, of protein kinase the as Ang II to induce the plasma membrane recruitment of NKA molecules C.E. Efendiev R. Bertorello A.M. Pedemonte C.H. J. Pharmacol. 2002; PubMed Scopus (30) Google Scholar). with this the of Tyr-255 for phenylalanine in NKA α1 the stimulation of NKA activity by either or To determine the effect of the α1 the Ang plasma membrane recruitment of NKA molecules, we the plasma membrane of NKA molecules in cells that have or not with Ang we used the of plasma membrane protein in of (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, R. Bertorello A.M. Pressley T.A. Rousselot M. Feraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (84) Google Scholar, R. J. Leibiger B. Katz A.I. Berggren P.O. Pedemonte C.H. Leibiger I.B. Bertorello A.M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). After treatment of the cells with either or Ang II, the cells were transferred to a the at the plasma membrane proteins were with The low the and the intracellular of NKA molecules. in NKA activity in response to AT1Rs activation are by in of NKA at the plasma membrane the in cells expressing the α1 stimulation of AT1Rs to increase NKA at the plasma of NKA molecules at the plasma membrane determined in cells expressing the NKA WT α1 and the α1 mutant were not that NKA was at the level in cell We the sequence the binding site by at with NKA and determined by stimulation of AT1Rs the association of NKA and molecules in cells expressing the wild-type α1, this effect was greatly in cells expressing the α1 mutant used for IP, from the cell were by and by with α1 antibody not The amount of NKA α1 was determined in the cell to cell and all treatment Thus, the in the are not due to different of NKA for as in of NKA α1 were from cells expressing WT α1 and the mutant. In cells expressing the NKA α1 the antibody recognition for is not different from the level for the performed with serum or antibody type as the α1 in suggest that treatment of the cells with Ang II the association of with NKA molecules and that this association is in cells expressing the NKA α1 mutant. results are with in 1 and We have previously demonstrated that stimulation of AT1Rs induces the phosphorylation of Ser-11 and Ser-18 of NKA α1 and that of these the Ang II-dependent regulation of NKA activity (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Pedemonte C.H. Efendiev R. Bertorello A.M. Semin. Nephrol. 2005; 25: 322-327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). with that the Ang II-dependent of NKA and is by phosphorylation of NKA The of difference in phosphorylation level between cells expressing WT and mutant α1 that the of recruitment of NKA α1 of for the between NKA and molecules was obtained between fluorophores attached to the two at a is the to protein-protein We have previously the of this R. Cinelli A.R. Leibiger I.B. Bertorello A.M. Pedemonte C.H. 2006; PubMed Scopus Google Scholar). After treatment with Ang II the cells were to polyclonal rabbit and monoclonal mouse antibodies NKA α1 and AP-1, the cells were with Cy3-tagged anti-rabbit and Alexa Fluor 488-tagged anti-mouse and Alexa Fluor are for determinations L. S. R.A. PubMed Scopus Google Scholar, A. B. J. Cell 2003; PubMed Scopus Google Scholar, J.S. S. A. A. E. Am. J. Physiol. 2006; 290: PubMed Scopus Google Scholar). in demonstrate as as of the cells obtained in the Alexa Fluor and determinations of normalized FRET, of cells with of the fluorophores were not FRETN, is of the concentrations J. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (50) Google was determined through and is in 2 as In NKA α1 is in the plasma membrane and is in the We have previously demonstrated that the intracellular compartments containing NKA molecules are to the plasma membrane and that is to between NKA molecules that are at the plasma membrane and in intracellular compartments R. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Pharmacol. PubMed Scopus (21) Google Scholar). In cells with Ang II, that molecules have to the plasma membrane the intracellular compartments are and the level of between the fluorophores attached to NKA and In cells in the Ang II-dependent phosphorylation of NKA α1 by with the protein kinase C chelerythrine chloride, the level of was greatly a some of the cells were with the to Ang II, induces the of NKA molecules T. Lokhandwala M.F. Clin. Exp. 1997; PubMed Scopus Google Scholar, T. Lokhandwala M.F. Am. J. Physiol. 1997; Google and level of was of were performed in cells expressing the and mutants of Ang II stimulation of AT1Rs level of between the fluorophores attached to NKA α1 and in cells expressing the not in cells expressing the mutant of a protein with is a in the recruitment of proteins to the plasma membrane H. J. H. S. T. A. T. J.S. 1995; PubMed Scopus Google Scholar). In this we present evidence that the sequence within the NKA α1 is the site of binding of in response to In of this we present results obtained by different and methods and The Ang II-dependent stimulation of NKA activity was not due to a expression of the α1 mutant. the level of expression of NKA α1 is the in cells expressing the wild-type α1 and the α1 mutant is by the of the level of NKA NKA at the plasma membrane, and binding to may with motifs with the sequence is a residue and a residue T. Rev. Cell Biol. 1999; PubMed Scopus Google Scholar). The NKA α1 have two of these and We have previously demonstrated that PMA, activates the NKA activity through the as Ang II (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google stimulates the NKA containing the WT 1 at the level as NKA molecules containing the mutants and (20Done S.C. Leibiger I.B. Efendiev R. Katz A.I. Leibiger B. Berggren P.O. Pedemonte C.H. Bertorello A.M. J. Biol. Chem. 2002; 277: 17108-17111Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). the NKA α1 sequence is the site of binding that is to the Ang II-dependent regulation of NKA activity. Cells expressing the WT and mutant forms of NKA α1 were and maintained all the time in 3 μm ouabain the expression of the endogenous α1-subunit C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (71) Google Scholar, C.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, endogenous α1 not have with determinations performed in cells expressing the mutants of We have previously demonstrated as of the mechanism of of NKA molecules by stimulation of interacts with the sequence within NKA α1 (20Done S.C. Leibiger I.B. Efendiev R. Katz A.I. Leibiger B. Berggren P.O. Pedemonte C.H. Bertorello A.M. J. Biol. Chem. 2002; 277: 17108-17111Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We determined that the effect Ang II-dependent stimulation of NKA activity and in the NKA determined by to the effect of Ang II, stimulation of produced of NKA not increase the of NKA α1 with AP-1. as as the that the Ang phosphorylation of NKA α1 the NKA interaction, the that the NKA is a response to the activation of Ang II-dependent plasma membrane recruitment of NKA molecules requires the protein kinase phosphorylation of NKA α1 (16Efendiev R. Budu C.E. Cinelli A.R. Bertorello A.M. Pedemonte C.H. J. Biol. Chem. 2003; 278: 28719-28726Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The that the Ang II-dependent of was the cells were with the protein kinase C chelerythrine chloride is evidence of the between the phosphorylation of α1 and the The results of these experiments suggest that the Ang II-dependent is not due to some as membrane or protein to the plasma that the effect of Ang II is effect produced in a Thus, the are not due to the of proteins to the plasma AP-1, through interacts with the cargo H. H. J.S. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. T. Y. R.C. H. T. T. J.S. 1999; PubMed Scopus Google Scholar). Although containing the in the traffic between the and containing the to in cells and to responsible for of proteins to the basolateral membrane H. H. J.S. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. T. Y. R.C. H. T. T. J.S. 1999; PubMed Scopus Google Scholar). Although we have not is that the is in is from proximal tubule the regulation of NKA by Ang II in a and not protein traffic through the that containing the responsible for the results described in this vesicle-dependent traffic of membrane proteins requires the of the protein cargo with T. Rev. Cell Biol. 1999; PubMed Scopus Google Scholar). are and have a within the cell by to the NKA α1 sequence during plasma membrane (20Done S.C. Leibiger I.B. Efendiev R. Katz A.I. Leibiger B. Berggren P.O. Pedemonte C.H. Bertorello A.M. J. Biol. Chem. 2002; 277: 17108-17111Abstract Full Text Full Text PDF PubMed Scopus (62) Google in this we present evidence that Ang traffic of NKA molecules requires the binding of to sequence at Tyr-255 within the NKA demonstrate the of two that the NKA molecules traffic from or to the plasma membrane in response to G-protein-coupled receptor We Dr. for the fluorescence