Dopamine-induced Endocytosis of Na+,K+-ATPase Is Initiated by Phosphorylation of Ser-18 in the Rat α Subunit and Is Responsible for the Decreased Activity in Epithelial Cells

Abstract

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the α subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat α1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat α1subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased α subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type α1 subunit or the S11A α1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A α1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affectper se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased α subunit phosphorylation induced by this agonist.We conclude that dopamine-induced inhibition of Na+,K+-ATPase activity in rat renal tubule cells requires endocytosis of the α subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process. Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the α subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat α1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat α1subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased α subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type α1 subunit or the S11A α1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A α1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affectper se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased α subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+,K+-ATPase activity in rat renal tubule cells requires endocytosis of the α subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process. clathrin-coated vesicles G protein-coupled receptors protein kinase C proximal convoluted tubules opossum kidney bisindolylmaleimide dopamine wild type early endosomes late endosomes 2-(N-morpholino)ethanesulfonic acid. Regulation of the Na+ channel (1Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), H+,K+-ATPase (2Courtois-Coutry N.D. Roush D. Rajendran V. McCarthy J.B. Geibel J. Kashgarian M. Caplan M. Cell. 1997; 90: 501-510Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and Na+,K+-ATPase (3Schmalzing G. Eckard P. Kröner S. Passow H. Am. J. Physiol. 1990; 258: C179-C184Crossref PubMed Google Scholar, 4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar) activity appears to involve removal from, or retention within, the plasma membrane of these transport proteins: while Na+ channels and H+,K+-ATPase activity in intact cells are constitutively increased by the removal of specific endocytic signals, internalization of Na+,K+-ATPase is associated with activation of specific membrane receptors and depends on the sequence of intracellular signals generated from this interaction (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar,5Bertorello A.M. Katz A.I. Am. J. Physiol. 1993; 265: F743-F755PubMed Google Scholar). Endocytosis of integral membrane proteins is initiated by selective recognition of the target protein followed by its transport, usually via clathrin-coated vesicles (CCV),1into endosomes (6Pierce B.M. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar, 7Lamaze C. Schmid S.L. Curr. Opin. Cell Biol. 1995; 7: 573-580Crossref PubMed Scopus (254) Google Scholar, 8Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (557) Google Scholar, 9Kirchhausen T. Bonifacino J.S. Reizman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (352) Google Scholar). While endocytosis of membrane receptors as part of their signaling mechanism has been extensively studied, internalization of integral membrane proteins triggered by a network of signals generated after activation of G proteins-coupled receptors (GPCRs) is less well documented. Inhibition of Na+,K+-ATPase activity by activation of GPCRs (dopaminergic) in renal epithelial cells is associated with the removal of active units from the basolateral plasma membrane and their transport, via clathrin-coated vesicles, into early (EE) and late endosomes (LE) (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). This process requires activation of phosphatidylinositol 3-kinase (10Chibalin A.V. Zierath J. Katz A.I. Berggren P.-O. Bertorello A.M. Mol. Biol. Cell. 1998; 9: 1209-1220Crossref PubMed Scopus (90) Google Scholar) and is dependent on protein kinase C (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Protein kinase C (PKC) phosphorylates the Na+,K+-ATPase α1 subunit in cell-free preparations (12Lowndes J.M. Hokin-Neaverson M. Bertics P.J. Biochim. Biophys. Acta. 1990; 1052: 143-151Crossref PubMed Scopus (87) Google Scholar, 13Bertorello A.M. Aperia A. Walaas I. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11359-11362Crossref PubMed Scopus (289) Google Scholar, 14Chibalin A.V. Vasilets L.A. Hennekes H. Pralong D. Geering K. J. Biol. Chem. 1992; 267: 22378-22384Abstract Full Text PDF PubMed Google Scholar, 15Nishi A. Bertorello A.M. Aperia A. Acta Physiol. Scand. 1993; 149: 377-384Crossref PubMed Scopus (14) Google Scholar, 16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and this event is associated with a decrease in its catalytic activity (13Bertorello A.M. Aperia A. Walaas I. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11359-11362Crossref PubMed Scopus (289) Google Scholar, 15Nishi A. Bertorello A.M. Aperia A. Acta Physiol. Scand. 1993; 149: 377-384Crossref PubMed Scopus (14) Google Scholar). However, because in some reports investigators have failed to demonstrate an effect of PKC on the hydrolytic activity of purified preparations (16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 17Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1994; 269: 30436-30444Abstract Full Text PDF PubMed Google Scholar), or of phorbol esters in cell lines (18Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1997; 272: 17726-17733Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), it remains unclear whether Ser/Thr phosphorylation of the α1 subunit represents a physiological mechanism responsible for Na+,K+-ATPase inhibition in intact cells. In PCT (proximal convoluted tubule) segments phorbol esters (19Bertorello A.M. Aperia A. Am. J. Physiol. 1989; 256: F370-F373PubMed Google Scholar, 20Ominato M. Satoh T. Katz A.I. J. Membr. Biol. 1996; 152: 235-243Crossref PubMed Scopus (120) Google Scholar) and diacylglycerol analogs (20Ominato M. Satoh T. Katz A.I. J. Membr. Biol. 1996; 152: 235-243Crossref PubMed Scopus (120) Google Scholar, 21Bertorello A.M. J. Cell Sci. 1992; 101: 343-347Crossref PubMed Google Scholar) inhibit Na+,K+-ATPase activity. In OK (opossum kidney) cells and Xenopus oocytes phorbol esters decrease ouabain-sensitive Rb+ transport, and this decrease is associated with phosphorylation of the Na+,K+-ATPase α1 subunit (22Middleton J.P. Khan W.A. Collingsworth G. Hannun Y.A. Medford R.M. J. Biol. Chem. 1993; 268: 15958-15962Abstract Full Text PDF PubMed Google Scholar,23Vasilets L.A. Cell. Physiol. Biochem. 1997; 7: 1-18Crossref Scopus (20) Google Scholar). However, expression of a mutated form (Ser-18) alone or in combination with another mutation (Ser-11) in Xenopusoocytes revealed a variable response of Rb+ transport, depending on whether phorbol esters or purified PKC was used (23Vasilets L.A. Cell. Physiol. Biochem. 1997; 7: 1-18Crossref Scopus (20) Google Scholar). Moreover, phorbol esters have been also associated with stimulation of ouabain-sensitive Rb+ transport in PCT segments (24Feraille E. Carranza M.-L. Buffin-Meyer B. Rousselot M. Doucet A. Favre H. Am. J. Physiol. 1995; 268: C1277-C1283Crossref PubMed Google Scholar, 25Carranza M.-L. Féraille E. Favre H. Am. J. Physiol. 1996; 271: C136-C143Crossref PubMed Google Scholar) and OK cells (26Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (38) Google Scholar). Earlier experimental approaches often relied on the action of phorbol esters as direct activators of protein kinase C. of Na+,K+-ATPase to these in intact cells to phosphorylation of V. A. I. and A. M. for in increased endocytosis J. I. P. Geering K. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar) that Na+,K+-ATPase a we have that inhibition of Na+,K+-ATPase activity in renal proximal tubule cells was associated with phosphorylation and selective endocytosis of the α1 subunit (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar, A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). these two events were because the rat α1 subunit an that the PKC target was not in response to dopamine (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). While these that phosphorylation of the α subunit in response to dopamine is necessary for it remains unclear whether inhibition of the enzymatic activity in intact cells from phosphorylation of the subunit in the plasma membrane that Na+,K+-ATPase a in that decrease its or from the endocytic inhibition as a of Na+,K+-ATPase units in the plasma In because the two residues (Ser-11 and Ser-18) by PKC in purified preparations (16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) are the of the α1 subunit, in this we have site-directed mutagenesis to in intact cells whether or or both are phosphorylation in Na+,K+-ATPase endocytosis and inhibition of its catalytic activity in response to were from bisindolylmaleimide and were from Na+,K+-ATPase α subunit in vesicles and early and late endosomes was a by M. Caplan of the Na+,K+-ATPase in the phosphorylation was performed a the rat Na+,K+-ATPase α subunit M.-L. Féraille E. Favre H. Am. J. Physiol. 1996; 271: C136-C143Crossref PubMed Google Scholar). PCT cells were as (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). and were the were and the the was on to a was with in of was for the being to and was by the on and to a cell PCT cells were to by for in to the cells and of and were on were to a protein of and were used after type and forms of the Na+,K+-ATPase α1 subunit of rat were from for expression by cells in type α1 was a of B. J. J.B. PubMed Scopus Google Scholar). type sequence was with the and to of with followed by of the wild type with were into for and of an for the α1 was with the and its of an for the was with the and its was for both of the was by with the and by of the were from the early of in OK cells were with the (26Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (38) Google Scholar, 1991; Google Scholar). after the cells were to a the Na+,K+-ATPase of OK cells is to this of OK cells that the Na+,K+-ATPase the α subunit to were and the in were performed with a of cells in were in in the or of for was by the on Cell of were to the Na+,K+-ATPase in and specific activity, in Na+,K+-ATPase activity was after of the cells as (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). Na+,K+-ATPase activity was in cells to this after the activity was in a these two represents the activity of the isoform. of Na+,K+-ATPase is in intracellular of their of of in and of cell activity, of of (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). their in these compartments the of in we have by the Na+,K+-ATPase hydrolytic activity in PCT cells by to a well for cell with I. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In Na+,K+-ATPase activity of of in PCT cells to was not from that of PCT cells by with cells to for were with also the of the the Na+,K+-ATPase activity was increased This to a the of activity in these and that of intact cells or with alone in of the plasma membrane not of from intracellular as vesicles and early PCT cells of were in a and with the of OK cells of were in the for with were performed was by the of and and the on cells were by of the Na+,K+-ATPase α subunit was performed as (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). of were with of that this does not the wild type α and the of protein were by the PubMed Scopus Google Scholar). were to and to were by and was performed as (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). In to the of the α subunit phosphorylation we used the E. Greengard P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). PCT cells were in the or of dopamine for was by of and of the α subunit was performed as α subunit was by purified protein kinase C in the of were by were by into the α subunit from the was and the of of of α subunit was as (13Bertorello A.M. Aperia A. Walaas I. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11359-11362Crossref PubMed Scopus (289) Google Scholar). of α subunit was by as OK cells in of were was by the to and of and were to of the a and the were to a were on a as (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar), the of P. M. J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). were not was in was in (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). of vesicles was performed as (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar, Am. J. Physiol. 1994; PubMed Google Scholar). with or OK cells were a in and to with was for and the was in the and to a and were for and from the were in and for was to a of of protein and was for were for both and (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). of early and late another was the and to cell and plasma plasma were purified as (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Protein was to Biochem. Scopus Google Scholar). were with an or were performed a of the Na+,K+-ATPase α subunit was performed as (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) a and the were a two experimental were by the of with was was Na+,K+-ATPase α subunit is in response to dopamine, phosphorylation being after with phosphorylation we have in two that dopamine the of subunit of and the of Na+,K+-ATPase α subunit was the the that the α subunit is in the of α subunit from cells was to a of that from cells to This that in intact cells dopamine phosphorylates the Na+,K+-ATPase α subunit with a of of To the of α subunit phosphorylation and internalization inhibition of Na+,K+-ATPase activity in PCT we the effect of dopamine on Na+,K+-ATPase activity and α subunit phosphorylation endocytosis was We have that with wortmannin or LY294009 the endocytosis of α into vesicles and early and late endosomes induced by dopamine (10Chibalin A.V. Zierath J. Katz A.I. Berggren P.-O. Bertorello A.M. Mol. Biol. Cell. 1998; 9: 1209-1220Crossref PubMed Scopus (90) Google Scholar). In the we the effect of dopamine on Na+,K+-ATPase activity and α subunit phosphorylation in PCT cells that have been with wortmannin or for with the Dopamine alone increased the of phosphorylation of the α subunit from the basolateral membrane and Na+,K+-ATPase activity phosphorylation was not by wortmannin or LY294009 However, the inhibition of Na+,K+-ATPase activity was blocked by with wortmannin or LY294009 or LY294009 alone did not Na+,K+-ATPase activity or α subunit that phosphorylation is a not a event for Na+,K+-ATPase it the endocytosis, it is the removal of the from the plasma the increased se that Na+,K+-ATPase activity in intact cells. To the of PKC phosphorylation residues inhibition of Na+,K+-ATPase activity by dopamine, OK cells were with of the rat α1 subunit in or Ser-18 were by OK cells expressing wild type and the S11A and S18A α1 subunit expressed of α1 as by and the Na+,K+-ATPase activity in plasma was also In intact OK cells dopamine Na+,K+-ATPase activity and this inhibition was by with In OK cells expressing the S11A the inhibitory effect of dopamine was less that in cells expressing the it was This effect as well was by In contrast, dopamine failed to a in Na+,K+-ATPase activity in cells expressing the S18A has been T. B. I. Nairn A.C. E. Greengard P. Aperia A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) that cells expressing the Na+,K+-ATPase α1 a mutation in a protein kinase C phosphorylation to Ser-18 in to to or We have whether OK cells on a of and and in in any that after of OK cell and and and we have not the intracellular or of the OK cells did not any in cell or an that a in cell to an intracellular that the these two the cell lines used of the by protein kinase C. To the of α1 subunit phosphorylation OK cells were with and with or dopamine of phosphorylation of the α1 subunit was by and for the of by Dopamine increased the α1 subunit phosphorylation in cells as well as in OK cells expressing the S11A mutant. in phosphorylation was in and S11A and both effects were blocked by dopamine failed to the α1 subunit in OK cells expressing the S18A mutant. of 4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar) inhibition of Na+,K+-ATPase activity is the of a of units the plasma membrane and intracellular we in from it is that with dopamine a of α1 that the plasma in the the of α1 in does not While the of cell on is to the of these two it does not the of the specific into the are to the of the α and the in are we whether phosphorylation of Ser-18 is essential for endocytosis by the of α1 in early and late OK cells and in were with or dopamine for from that used to phosphorylation this was to to an of α1 subunit in both early and late endosomes 4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Dopamine increased the of α1 in early and late endosomes in and to a in early endosomes from S11A this effect was blocked in both by with the PKC alone effect on α1 subunit However, in OK cells expressing the Na+,K+-ATPase α1 subunit the S18A dopamine failed to a in α1 subunit in early or late endosomes This for a phosphorylation of Ser-18 and endocytosis of the α subunit in response to To whether phosphorylation of Ser-18 was also for clathrin-dependent internalization of the were from OK cells expressing the or S18A α1 subunit S11A was not in this because this mutation does not the of dopamine to the α subunit to inhibit its activity. OK cells in were with or While dopamine increased α1 subunit in from cells it failed to in OK cells the S18A of this demonstrate that inhibition of Na+,K+-ATPase activity in intact cells is by a of active units the plasma of the Na+,K+-ATPase α subunit in the basolateral plasma membrane the Ser-18 this event se does not the catalytic activity. the Na+,K+-ATPase activity in intact cells is by the removal of from the basolateral plasma membrane and internalization by endocytosis, for phosphorylation of the catalytic subunit appears to as a Dopamine increased the of Na+,K+-ATPase α subunit phosphorylation by In phosphorylation of the α subunit a of of PKC the α subunit from cells to a of and of from cells. we have that dopamine phosphorylates the Na+,K+-ATPase to a of of of α subunit, of of α subunit phosphorylation has been to and for the rat α subunit, in Ser-18 it has been to M.S. Sweadner K.J. J. Biol. Chem. 1997; 272: 17726-17733Abstract Full Text Full Text PDF PubMed Scopus (91) Google and While were performed purified preparations and protein kinase the in intact cells in response to a has not been We have that activation of GPCRs by dopamine inhibits Na+,K+-ATPase activity in renal epithelial cells and that this inhibition was associated with endocytosis of its α and (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). via and transport into early and late This process requires stimulation of phosphatidylinositol 3-kinase activity, the of with the increased of α in the increased of α in and was blocked by two phosphatidylinositol 3-kinase wortmannin or LY294009 (10Chibalin A.V. Zierath J. Katz A.I. Berggren P.-O. Bertorello A.M. Mol. Biol. Cell. 1998; 9: 1209-1220Crossref PubMed Scopus (90) Google Scholar), in their and of of these a for whether preventing endocytosis the of α subunit phosphorylation in the plasma membrane the in its catalytic activity in response to In this the of phosphorylation in the of dopamine was to that (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and was not by the of wortmannin or However, the of dopamine to inhibit Na+,K+-ATPase activity was PCT cells were with of the two phosphatidylinositol 3-kinase by did not the of phosphorylation or of endocytosis, that these two have a of dopamine to inhibit Na+,K+-ATPase activity endocytosis was is in with a we have that the inhibited the of Na+,K+-ATPase into and and that these dopamine failed to inhibit Na+,K+-ATPase activity (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). the α subunit was the and this dopamine inhibited Na+,K+-ATPase activity, that subunit internalization in is to a decrease in Na+,K+-ATPase activity. In these that it is the removal of the α subunit from the plasma membrane its phosphorylation that in cell Na+,K+-ATPase activity, a mechanism to that of transport proteins as the epithelial channel (1Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) and (2Courtois-Coutry N.D. Roush D. Rajendran V. McCarthy J.B. Geibel J. Kashgarian M. Caplan M. Cell. 1997; 90: 501-510Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). site-directed mutagenesis we also in this that endocytosis of Na+,K+-ATPase and inhibition of its catalytic activity in response to phosphorylation of the Ser-18 in the α1 subunit of another α1 subunit in by PKC (16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), did not the of to phosphorylation and internalization of the α1 subunit the inhibitory action of on the enzymatic activity. and Ser-18 residues of the Na+,K+-ATPase α1 subunit that phosphorylation have been identified in cell-free preparations by purified PKC (16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) or in intact cells after activation of PKC by phorbol esters (23Vasilets L.A. Cell. Physiol. Biochem. 1997; 7: 1-18Crossref Scopus (20) Google Scholar, I. Nairn A.C. M. Greengard P. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). that of the subunit to the was to (16Feschenko M.S. Sweadner K.J. J. Biol. Chem. 1995; 270: 14072-14077Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), a physiological is that in response to a physiological Ser-18 in the rat α1 subunit is and that this is essential for endocytosis of the α subunit and inhibition of Na+,K+-ATPase activity. of although a PKC cell-free preparations of did not its endocytosis in intact cells the in Na+,K+-ATPase activity by dopamine, that the does not a in this process in the is as by Vasilets (23Vasilets L.A. Cell. Physiol. Biochem. 1997; 7: 1-18Crossref Scopus (20) Google Scholar), that in Ser-18) represents the PKC phosphorylation that Na+,K+-ATPase activity and α1 In Na+,K+-ATPase activity in intact cells represents the of active units the basolateral plasma Na+,K+-ATPase activity in response to GPCRs signals in renal epithelial cells is by removal of the from the plasma membrane Ser-18 of the α subunit and this it does not in the of the the are as the increased of α and in intracellular compartments is not by a in Na+,K+-ATPase catalytic activity (4Chibalin A.V. Katz A.I. Berggren P.-O. Bertorello A.M. Am. J. Physiol. 1997; 273: C1458-C1465Crossref PubMed Google Scholar). some of the in late endosomes (11Chibalin A.V. Pedemonte C.H. Katz A.I. Feraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This a that as a for their to the plasma membrane of a has been in epithelial cells A.M. K. A.V. Katz A.I. Am. J. 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