The Role of the β1 Subunit of the Na,K-ATPase and Its Glycosylation in Cell-Cell Adhesion

Based on recent data showing that overexpression of the Na,K-ATPase β1 subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the β1 subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase α1 and β1 subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, β-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca2+ depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β1 subunit and the endogenous α1 subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous β1 subunits in the lateral membrane were substituted by unglycosylated β1 subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase β1 subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase β1 subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase β1 subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation. Based on recent data showing that overexpression of the Na,K-ATPase β1 subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the β1 subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase α1 and β1 subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, β-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca2+ depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β1 subunit and the endogenous α1 subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous β1 subunits in the lateral membrane were substituted by unglycosylated β1 subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase β1 subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase β1 subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase β1 subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation. Specialized tight epithelia of multicellular organisms function as barriers that maintain the distinct molecular composition of apical and basolateral plasma membrane domains. Individual cells within the monolayer are linked with each other to maintain its structural integrity and to retard or prevent the diffusion of solutes through the intercellular space. The junctional complex in epithelial cells consists of tight junctions, adherens junctions, and desmosomes (1Farquhar M.G. Palade G.E. J. Cell junctions and desmosomes cells, tight junctions are for intercellular junctions are important for cells of the monolayer by of and from cell to of cell junctions is a in the from cells to a disruption of junctions and structural contacts cells is the in the the process in which cells from a highly polarized epithelial to a Cell J. with and of J. J. contacts are also in J. J. and kidney J. of the essential for the or of cell junctions that of Na,K-ATPase by to or by depletion in cell in J. Cell J. or tight junction formation surface-attached Madin-Darby canine kidney (MDCK) Madin-Darby canine the the and the Na,K-ATPase β1 Madin-Darby canine the the and the Na,K-ATPase β1 cells in J. of the Na,K-ATPase also increased of polarized of epithelial cells and polarized cell line to and J. J. J. J. were as showing that of the Na,K-ATPase is important for formation and of cell-cell contacts. the Na,K-ATPase to intercellular adhesion is was that the pump of the that is involved in formation J. the Na,K-ATPase is important for of of the normal membrane and normal of and other and cell adhesion on of The that on cell adhesion were to the detected upon of cells or in the of the that increased of J. that the of the by the Na,K-ATPase is for cell-cell of the Na,K-ATPase β1 subunit in nonpolarized cells and MDCK cells by increased cell-cell adhesion in cell J. data that the Na,K-ATPase β1 subunit as an in cell overexpression of the β1 subunit increased of the endogenous α1 subunit J. J. data be is that of by cell is a of of the increased Na,K-ATPase to cell adhesion or a role of the α1 that overexpression of the Na,K-ATPase β1 subunit in cells cell to the subunits of the and the that the Na,K-ATPase β1 subunit is involved in cell-cell adhesion by a structural cells, we association of the pump with of the junctional complex of the monolayer and after disruption of cell to cell contacts. In we the of of from the β1 subunit on its to adherens junctions. we the of of MDCK cells to a β1 antibody and to on cell contact formation. The of that the Na,K-ATPase with the adherens junctions upon the of cell junction formation. disruption of cell the Na,K-ATPase is from the of cell contact with adherens junction of from the Na,K-ATPase β1 subunit association of the with the adherens junctions and the of cell-cell of a antibody to the extracellular domain of the Na,K-ATPase β1 subunit cell-cell contact to data that the Na,K-ATPase β1 subunit plays an important role in the of contacts MDCK cells, an important in the of a tight of of the Na,K-ATPase β1 and the of linked to the of the Na,K-ATPase β1 subunit was as J. or were by the The a of the and was by cells were from In to cell or MDCK cells were on and with the or the cell were by after the a of of was were and in the of of of in with the of or were and for of the of of or or were for after on or were the were for and in cell-cell adhesion antibody against a antibody against the Na,K-ATPase α1 subunit a antibody against a antibody against a antibody against a antibody against the of a antibody against the Na,K-ATPase β1 and the antibody against and that also and a antibody against of Cell were by with in for or after treatment with as and by with for cells were with for was by The was antibody against of other was by a with the by a with of the or or or of MDCK treatment of MDCK cells was by a J. Cell cells on or were with Ca2+ and and with the for cells were in by with for by and to the or to of and of the Na,K-ATPase and MDCK cells or MDCK cells or β1 subunits were for after in in of the apical or membrane was by J. J. Cell were with that was from the apical or basolateral the cells were and by with of in with and Cell were by of with of in were to from the cells by apical or basolateral the of the cell was with of in a of of in with and with for The were on the and were from the by in of for and by with the from the cells. cells on the were with in for after and cell by were a membrane and by the the antibody and the to as a antibody to the were by MDCK cells or cells a or were and on a in the cells were by The of the cells of on the were cells or of were this a or were the were the adhesion was by of the on of cell with or the the or adhesion was by the of cells that form contacts with the cells the of The against the Na,K-ATPase β1 or were from and other molecular and to to their in a cell-cell adhesion and after of the was The antibody against the Na,K-ATPase β1 subunit was of or in the cell medium. The in of the Na,K-ATPase β1 subunit was to the a or with of the antibody against the Na,K-ATPase β1 subunit after a of the and antibody in medium. The against and were the of for a internalization of the antibody against of the antibody were after and of cell was of or Na,K-ATPase to endogenous Na,K-ATPase α1 and β1 subunits were detected by in the basolateral plasma membrane domain of MDCK cells and that were by the was by an antibody against a protein, The to was detected in the and was detected in the and were by that treatment of cells with the cytoskeleton and to the all and membrane that are linked to the cytoskeleton J. Cell J. Cell In with treatment of MDCK cells with to the J. Cell the in the cell that were from cells by the In contrast, α1 and β1 subunits from the basolateral the Na,K-ATPase subunits in the basolateral plasma membrane domain are to detergent that the is or with the cytoskeleton the basolateral In contrast, the of the Na,K-ATPase subunits was to the detergent The cell Na,K-ATPase subunits The β1 subunit was by in the of the was in the basolateral membrane and the to the form of the β1 the by on a of the that in the The from the cells with also the of the α1 and β1 subunits were and the β1 subunit was detected as a that the of the pump of the α1 subunit with the of the β1 subunit was by the detected by antibody in the cell in the to the cell after treatment of cells that with the antibody were by to the endogenous Na,K-ATPase the expressed was also detected on the basolateral membrane The basolateral of was also to of the detergent of the endogenous Na,K-ATPase α1 or β1 as detected by the of the the antibody against the β1 subunit and the of the with the antibody against the α1 subunit was detected in the apical and basolateral of to the by was also by of MDCK cells with from the lateral and of the Na,K-ATPase to detergent that the is or with the cytoskeleton the basolateral that the Na,K-ATPase α1 subunit with the membrane protein, a protein, in cell that this is for the of the Na,K-ATPase in MDCK cells to of the pump the basolateral plasma membrane domain J. Cell J. Cell In with of the tight monolayer of MDCK cells against of with in the lateral membrane was also detected as a the cells in to that was from the that other association are involved in the of the Na,K-ATPase on the lateral in MDCK cells. The Na,K-ATPase the of Cell in the of MDCK from the of MDCK cells, is in the lateral and is from the membrane that the contacts with the cells are important for stable of the in the lateral from the of of the cell of in the lateral is the of a of The is also in a of the cells was after treatment of cells with this is a of membrane junctions of the cells, we cells in is that Ca2+ is for formation and of cell-cell contacts. of Ca2+ from the in of the lateral of cells and a in a lateral membrane as a line and as that the of the in the lateral is to the of cell-to-cell contacts. The Na,K-ATPase co-localized with cells to each other in the tight monolayer by formation of the tight junctions and adherens junctions. of MDCK cells an antibody against the tight junction protein, that with the tight junctions in the lateral membrane In contrast, displayed with a of adherens junctions, β-catenin, as detected by junctions are formed as a of the extracellular of the that in the lateral of cells is with adherens junctions the lateral membrane and with a to the domain of J. The complex of and with the cytoskeleton to of with that in to or other to this cytoskeleton the complex is to by J. of with was after cell treatment with in all of the cells the tight junctions. The and the of the cells are in In the of the cells, also was co-localized with in the the of contact cells the of MDCK cells and after by that the endogenous Na,K-ATPase α1 subunit was also to detergent and co-localized with the Na,K-ATPase with adherens junctions in the tight monolayer of MDCK cells. of the Na,K-ATPase with adherens junctions is after the detergent extraction. or association with adherens junctions is a of basolateral membrane protein, we expressed the basolateral as an in MDCK cells. of in the tight monolayer of MDCK cells was to that of in to was to the treatment of cells with The of was by the The of the that was in the cells after was co-localized with the as in the of is with the adherens junctions and of the of the Na,K-ATPase with cell junctions. the in of that formation of cell contacts and from a MDCK cell to a was detected in the membrane of cells and was from the of the plasma membrane In that or cells, in the formed lateral cells of cell contact we MDCK cells were on the and in the in the of cell contacts was the by of the of of cells in a are on of in the of cell contact as as contacts were The of in the of cell contact cells and of in a formed cell contact cells that to each other The in formed lateral with adherens junction and also co-localized with in the of cell contacts with the in the of the cells and this of formation of a the tight junctions were as from the of the tight junctional protein, the association of with the adherens junctions and cytoskeleton the stages of the of the cell The was the of the Na,K-ATPase upon cell is that adhesion as β-catenin, and are from the plasma membrane of polarized cells from each other and from the upon of Ca2+ from the or upon the of of In with internalization of in cells was detected after of cells in for was also and was partially co-localized with and that are from the lateral membrane as a precisely with the endogenous Na,K-ATPase α1 subunit the of Na,K-ATPase upon cell and with the of the of adherens junctions, data showing internalization of Na,K-ATPase subunits upon cell in are with the in which Na,K-ATPase α1 subunits J. and β1 subunits were after cell treatment with which was to cell that of the Na,K-ATPase from the lateral membrane is a of the cell process that is of the that cell In the Na,K-ATPase is co-localized with adherens junctions in polarized tight of MDCK cells. The with adherens junction after of the cells. The association of the Na,K-ATPase with adherens junctions with the formation of cell contact. disruption of cell the Na,K-ATPase is with adherens junction of MDCK by a against the of the Na,K-ATPase β1 the Na,K-ATPase β1 subunit is involved in cell-cell the antibody against of the Na,K-ATPase β1 subunit was as a The antibody the and lateral of MDCK cells was detected by of cells to the antibody after of cells in the of the antibody in the for of the antibody with the of the Na,K-ATPase β1 to the antibody to the of the cells as detected by the of The antibody against the extracellular domain of was as a in cell-cell adhesion this antibody to specifically intercellular adhesion of MDCK cells J. to the and lateral of MDCK cells a the antibody against was this antibody with on the of the cells specifically the adhesion cells. of cells a lateral membrane for cell-cell adhesion of formation of cell contacts in by we the cells to the of the antibody on cell-cell antibody to the extracellular domain of the endogenous canine β1 subunit of MDCK cells the β1 subunit and with the extracellular domain of that the β1 of we a basolateral protein, as a of the lateral in the on the of the antibody and other on cell-cell we MDCK cells and cell-cell adhesion by MDCK cells and to cell-cell adhesion that of the the of the adhesion cells were to the contacts with each other cells were The cells in the of the against and the Na,K-ATPase β1 subunit for displayed a of intercellular adhesion as with the The of cells that stayed was after and of cell in the of The of cells after with antibody was The of the antibody against the β1 subunit on all cell adhesion was and its a of of the antibody with the of the Na,K-ATPase β1 subunit the of cell adhesion The of the subunit antibody on cell adhesion of was from that with antibody against of the antibody to the extracellular domain of the Na,K-ATPase β1 subunit inhibited cell adhesion of MDCK cells to each other to the of the antibody J. of the intercellular adhesion of MDCK cells by the against and the Na,K-ATPase β1 or to surface-attached MDCK of cells that to the cells after of adhesion antibody against with the as by with the as by against the Na,K-ATPase β1 subunit against the Na,K-ATPase β1 subunit with the as by with the as by against the Na,K-ATPase β1 subunit with the as by with the as by against the Na,K-ATPase β1 subunit with the as by with the as by against the Na,K-ATPase β1 subunit against The of adhesion with the as by in a the that the antibody against the β1 subunit contacts cells to its to the β1 subunit to in the intercellular the on cell adhesion of the antibody against a a basolateral that is in cell-cell was The antibody that to the extracellular domain of the endogenous in MDCK cells was antibody lateral in the monolayer of MDCK cells with data In MDCK cells, the antibody and lateral of MDCK cells in a as with the antibody against the Na,K-ATPase β1 subunit and is that the the plasma membrane and in MDCK cells J. Cell decreased the of the antibody to the we cells in the of the antibody in the for the cells, and to the The of was decreased that the antibody was in the in and its to the to the for antibody maintain the of the antibody in the cell we the cell by of the antibody the adhesion The of cells after and of with antibody was to that The the that the antibody cell-cell adhesion by the extracellular domain of that be in to the extracellular domain of the Na,K-ATPase β1 this were the the antibody of the to the extracellular domain of in MDCK cells. of MDCK cells antibody in a with antibody detected on the lateral The of a of the antibody with the antibody the of a a antibody against the β1 subunit with the antibody even the antibody was that were of the antibody that the antibody with the antibody to the extracellular domain of the the of the antibody on cell adhesion is to its to the extracellular domain of the Na,K-ATPase β1 cell-cell adhesion Na,K-ATPase the of cell contact we the of the We a cell-cell adhesion in the of of and to the of the that were by other in to cell-cell J. We that cell-cell adhesion for the for the of which of cells after The the cells and the cells with after on the of the that the cells to from each other and the of in the of the all cells were of the of from of the which with J. data that Na,K-ATPase is essential for the of cell-cell the of contacts cells in is to of the extracellular domain of the β1 subunit by the antibody and to of of the to the Na,K-ATPase β1 for of the with the of the in the β1 subunit was by of with and were were expressed in MDCK cells. of cell the in molecular of the to The of of glycosylation on the of in MDCK cells was The was detected in the lateral in MDCK cells of or in of and the of glycosylation in the β1 subunit to be to lateral of the The of all in a and of the the lateral membrane and the β1 subunit all glycosylation the ability to with the α1 subunit and an heterodimer expressed in cells J. the in which the glycosylation is we the cells and by against a of is a to the and is essential for formed to from the plasma membrane The was partially co-localized with that the is in the membrane upon and in to In MDCK cells the the endogenous Na,K-ATPase α1 subunit was in the lateral to the or were co-localized with the endogenous Na,K-ATPase α1 subunit in the lateral membrane The was co-localized with the α1 subunit in the lateral membrane and in in to the membrane of the internalization of the endogenous Na,K-ATPase α1 The to the was to by The of was precisely co-localized with to the and was detected for the and In contrast, the was from the cells by detergent The of the was partially co-localized with that linked to the Na,K-ATPase β1 subunit are essential for stable association of the subunit with adherens junctions. In to the the was detected on the basolateral also on the apical membrane and The of the endogenous Na,K-ATPase β1 and α1 subunits in the apical that the of the in this the of the on the apical membrane and is an from the of the to the basolateral of the cell Further, that the is on the apical membrane the endogenous α1 of the in the basolateral membrane domain was by and and of the unglycosylated also decreased the of the endogenous α1 subunit to the detergent extraction. of the α1 subunit after the and is to of on to the the of the endogenous β1 subunit after the and in the Na,K-ATPase β1 subunit are essential for stable association of the Na,K-ATPase with the adherens junctions in MDCK cells. of the from the adherens junctions can the increased of the with the endogenous α1 subunit to and Further, of the glycosylation in and of this to the Na,K-ATPase β1 for glycosylation of the β1 subunit is important for cell-cell contact we the of in the cell-to-cell In to a stable MDCK cell line the for this was necessary to the of the expressed unglycosylated and endogenous β1 subunits in this cell We cell from cells and cells with the or unglycosylated β1 subunit by by a The of were the for all cell the as detected by the the of the was the antibody against the α1 and the was the antibody against the β1 subunit of the or unglycosylated decreased the of the endogenous β1 by and the of the endogenous α1 subunit was in the cells the and even increased in the cells the and is that the subunit of the Na,K-ATPase be expressed the subunit for normal from the and J. that the expressed or partially for the endogenous β1 subunits in the Na,K-ATPase we the apical and basolateral membrane from and cells. α1 subunit and of β1 subunit were detected in the apical in all cells. In the basolateral membrane the of of the endogenous Na,K-ATPase α1 subunit the of the endogenous β1 subunit decreased in cell the and β1 subunit by and and We that the on the lateral membrane also was in of this in MDCK cells in of a of the endogenous α1 subunit in that in the cell the pump is the and the plasma The of the Na,K-ATPase is by the α1 subunits with the In the basolateral half of the α1 subunits are with the endogenous β1 subunits and the other half with the unglycosylated the unglycosylated β1 subunit a complex with the α1 subunit J. the cell line the is an in which to the role of glycosylation in intercellular the of the in the basolateral membrane as cells, that this cell line is to maintain normal membrane and other of the the other the basolateral a of β1 subunits as with cells or cells the linked to the Na,K-ATPase β1 subunit are important for cell-cell the cells are to ability to form cell contacts. we a cell-cell adhesion for MDCK cells, cells the and cells The that the cell line unglycosylated displayed of cell-to-cell adhesion the cells and cells with The tight as cells contact with each and form tight junctions cells. of increased cell adhesion is essential for normal of the and is in of to the of cells to or to other of the role for the Na,K-ATPase β1 subunit in cell adhesion was by from and of that that of Na,K-ATPase β1 subunit is in cells with cell-cell as the cell J. the epithelia in kidney J. J. cells, cell cells, and cell from and J. J. J. In cells and the Na,K-ATPase α1 subunit with the subunit and in the apical J. J. J. data that the of the β1 subunit with the of the subunit be for apical of the the of the in the of cell and of intercellular in cell In the we that of MDCK cells with an antibody against the extracellular of the Na,K-ATPase β1 subunit specifically cell-cell adhesion to the of antibody J. that the of the antibody is to and to of the Na,K-ATPase that the extracellular domain of the Na,K-ATPase β1 subunit is involved in formation of cell contacts a structural cells. Further, we that linked to the β1 subunit are essential for cell-cell We cell-cell adhesion the stable MDCK cell line that expressed unglycosylated and that cell contact formation in this cell line as with cells or cells the by a we that the cell line a normal of the α1 subunit a in the of the endogenous β1 subunit in the lateral that half of the endogenous β1 subunits are substituted by unglycosylated subunits normal glycosylation of the β1 subunit is for intercellular The of normal of the β1 subunit for cell-cell adhesion is with data showing the of linked to the extracellular domain of the subunit in of the pump the of adherens junctions. We that the pump co-localized with adherens junctions their as cells to form a association was to treatment with and upon disruption of cell the Na,K-ATPase adherens junctions as were from the basolateral with data on lateral and detergent of the pump in MDCK cells J. Cell J. Cell J. Cell was that cell contact formation of cytoskeleton the of cell contact that in the Na,K-ATPase the lateral membrane J. Cell J. Cell the that the is to the Na,K-ATPase the of cell contact. The other that we is the of on the extracellular domain of the β1 We that the of a normal of the β1 subunit in of tight of the pump to the lateral membrane and adherens junctions, the pump can be easily from the lateral membrane by detergent and is is that the pump its ability to to the of on the extracellular domain of the β1 The β1 subunit a complex with the α1 subunit J. and to the plasma membrane and that the of the and the that in the domain of the Na,K-ATPase α1 subunit is that of to the cytoskeleton is to of the adherens junctional The the extracellular of the of cells is necessary to the Na,K-ATPase is in the lateral membrane by and extracellular as in and the as of the α1 subunit is linked to the β1 subunit the pump the of cell contact to with the β1 subunit of cells with the extracellular domain of or a that by to their is with recent data showing that the of adherens junctions, the of the Na,K-ATPase β1 subunit to epithelial and and of cells of the Na,K-ATPase β1 subunit in cells increased of in the plasma membrane as by which is with on and the β1 subunit the Na,K-ATPase β1 subunits of the cells also can the increased of and the adherens junctional complex in the lateral membrane to an support the of the Na,K-ATPase to the cytoskeleton that is in to The is also in with the recent that the β1 subunit an The subunit of the Na,K-ATPase of are with extracellular by for the β1 subunit and for the the subunit was as an adhesion on cells in the J. Cell The also is in with data on of the β1 subunit in cell J. MDCK cells with nonpolarized cells expressed the β1 subunit on the MDCK cells and on the MDCK and cells that a β1 subunit J. the β1 subunit was detected on the MDCK and cells cells were with the β1 The the β1 subunits of the cells can highly polarized of the Na,K-ATPase and in the of cell contact is with of cell-to-cell contact formation of the tight junctions that function as barriers the and apical plasma membrane and the Na,K-ATPase complex on the lateral on the in MDCK cells. The of that the of the Na,K-ATPase β1 subunit the lateral membrane of cells is essential for normal intercellular normal glycosylation of this subunit is for association with adherens junctions and of polarized The data the that of glycosylation of the subunit to the of or other in which of epithelia is to be We for the for We and for of the and with