Involvement of Galpha i2 in the Maintenance and Biogenesis of Epithelial Cell Tight Junctions*

Chandana SahaDagger , Sanjay K. Nigam§, and Bradley M. Denker

From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4- (activator of heterotrimeric G protein alpha  subunits) results in a 3-4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Galpha 0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753). Galpha i2 has been localized within the tight junction, and a role for Galpha i2 in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Galpha i2, or a GTPase-deficient mutant (constitutively activated), Q205Lalpha i2. Tryptic conformational analysis confirmed expression of a constitutively active Galpha i2 in Q205Lalpha i2-MDCK cells, and confocal microscopy showed a similar pattern of Galpha i2 localization in the three cell lines. Q205Lalpha i2-MDCK cells had significantly higher base-line TER values than wild type Galpha i2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Galpha i2); 377 ± 52 Omega ·cm2 (PC)), and both Galpha i2- and Q205Lalpha i2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4- during the Ca2+ switch had little effect on the kinetics of TER development in Galpha i2- or Q205Lalpha i2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4- (similar times to Galpha i2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4-. These findings indicate that Galpha i2 is important for both the maintenance and development of the TJ, although additional Galpha subunits are likely to play a role.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Polarized epithelia have developed highly specialized membrane functions enabling vectorial transport across the cellular layer. The junctional complex of epithelial cells includes gap junctions, adherens junctions, and tight junctions. The tight junction (TJ)1 is the most apical component of the junctional complex and provides two essential functions: (i) the permeability barrier to paracellular fluxes and (ii) the "fence" separating the apical and basolateral membrane domains. In developing tissues as well as cell culture models, the critical signaling events important to junction formation appear to be quite different from mechanisms that maintain junctional integrity. The TJ is composed of a complex of proteins that includes occludin, the only transmembrane protein identified so far (1). There are several peripherally attached membrane proteins found in the TJ including the zona occludens family (ZO-1, -2, and -3) (2-4). ZO proteins are members of the MAGUK (membrane associated guanylate kinase) superfamily that are often found at sites of cell-cell contact and may function to couple extracellular signaling pathways with the cytoskeleton. Other proteins found in or near the TJ include cingulin, 7H6, symplekin, unidentified phosphoproteins, and a series of signal transduction molecules (reviewed in Ref. 5).

MDCK cells are a cultured epithelial cell line that has been extensively utilized for studies of epithelial polarity, targeting of proteins, and the study of intercellular junctions (6). The Ca2+ switch model of TJ formation in MDCK cells has been widely utilized to gain insights into the function of polarized epithelial cells (7-11) and recapitulates many of the critical molecular events of epithelial morphogenesis. MDCK cells cultured in low calcium M) lack cell-cell contact, polarity, and junctions. "Switching" to normal calcium medium (NC) triggers a series of molecular events that leads to establishment of the polarized phenotype with characteristics of a tight transporting epithelium. Tight junction development can be followed by measuring the transepithelial resistance (TER), a rapid and reproducible assessment of tight junction integrity. Because MDCK cells are clonal and TJ development can be synchronized in the Ca2+ switch, the role of specific proteins on TJ biogenesis can be studied in this system by cDNA transfections.

The critical role of calcium in the formation of intercellular junctions is well established. Extracellular calcium is required for homotypic interactions of E-cadherin and is likely to be the initial event of junctional complex formation (12). Regulated intracellular calcium stores are also important for tight junction biogenesis. There are local increases in intracellular calcium concentration at the points of cell-cell contact (9), and chelation of intracellular calcium perturbs TER development (13). Thapsigargin depletes intracellular endoplasmic reticulum stores of calcium, and thapsigargin treatment of MDCK cells prior to initiation of cell-cell contact prevents TER development and the sorting of ZO-1 to the TJ (7). The signaling events important for TJ biogenesis are complex and utilize a variety of pathways. Phosphorylation events are important as several proteins become phosphorylated in the TJ, and protein kinase C (PKC) isoforms translocate to the TJ during biogenesis. PKC inhibitors markedly inhibit the development of TER in the calcium switch, and PKC agonists stimulate ZO-1 translocation to the membrane. The importance of PKC in tight junction biogenesis, as well as regulated calcium stores, suggests important roles for heterotrimeric G proteins. The proximity of several G proteins to the TJ also suggests they may have potential roles in regulating the development and/or maintenance of the TJ. PKCzeta and PKCalpha , have also been localized in the vicinity of the TJ (14-17). We previously demonstrated that expressing a constitutively activated Galpha o (Q205L) in MDCK cells significantly accelerated TJ biogenesis without affecting base-line resistances. Although Galpha o is a member of the G protein family inhibited by pertussis toxin (~80% similar to Galpha i1-3), its receptors and effectors are distinct, and furthermore, Galpha o is not detected in renal epithelia or MDCK cells (16, 18, 19). Several Galpha i family members are expressed in epithelial cells, and Galpha i2 has been shown to overlap with the tight junction in epithelial cell lines (16, 17). Taken together, these observations raise the possibility that Galpha i2 may be an important regulator of tight junctions. To test this hypothesis, we initially looked for effects of AlF4- (activator of Galpha subunits) on tight junctions in control cell lines and then established MDCK cell lines overexpressing wild type Galpha i2 and a constitutively activated Galpha i2 (GTPase-deficient, Q205Lalpha i2). We find that AlF4- significantly increases TER in control cells and accelerates TER development during the Ca2+ switch. The effects of AlF4- can be reproduced in MDCK cells expressing activated Galpha i2, indicating that this Galpha subunit is critical to the development and maintenance of tight junctions.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Plasmid Construction and Cell Culture-- Rat Galpha i2 cDNA was cloned into Bluescript (Stratagene) as described previously (20) and recloned into the EcoRI and ApaI sites of pcDNA3 (Invitrogen). Q205Lalpha i2 was provided by Dr. Gary Johnson and cloned into Bluescript using HindIII sites and then into pcDNA3 using XhoI and XbaI sites. MDCK cells were maintained in Dulbecco's modified Eagle's medium supplemented with antibiotics plus 5% fetal calf serum. Transfected cell lines were maintained in G418 (500 µg/ml; Life Technologies, Inc.)

Transfection-- Subconfluent MDCK cells (ATCC, Manassas, VA) were transfected with 10 µg of linearized plasmid by calcium phosphate precipitation method as described previously (16). G418-resistant colonies were analyzed for increased Galpha i2 expression by Western blot using a rabbit polyclonal antibody directed toward the C terminus of Galpha i2 (AS7; NEN Life Science Products). Control cells were obtained by transfecting pcDNA3 without insert, and all cell lines were established in parallel.

Tryptic Analysis of Transfected Clones-- Confluent PC-, Galpha i2-, or Q205Lalpha i2 -MDCK cells were washed twice with PBS and then scraped into buffer A (50 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 75 mM sucrose, 1 mM dithiothreitol, 1 mM EDTA). Cells were frozen and thawed three times and triturated ten times through a 27 gauge needle. All samples were incubated at 30 °C with no added nucleotide or 100 µM GTPgamma S. Samples were immediately placed on ice, and trypsin was added (20 pmol of L-1-(tosylamido)-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma). All samples were incubated at 30 °C for 20 min, and digestion was terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer followed by boiling for 5 min. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis and Western blot using AS7 anti-Galpha i2 rabbit polyclonal antibody (1:1,000) and ECL (Pierce) with goat anti-rabbit horseradish peroxidase (1:10,000).

Immunohistochemistry-- PC-, Galpha i2-, or Q205Lalpha i2-MDCK cells were grown on coverslips or Transwell filters (12 mm) (Costar), rinsed with PBS, and fixed with methanol (100%, -70 °C) for 10 min. Cells were then washed with PBS and blocked as described previously (16). Samples were incubated with rabbit polyclonal Galpha i2 (AS7, from NEN Life Science Products) at several dilutions and rat monoclonal to ZO-1 (undiluted supernatant; courtesy of D. Goodenough) for 1 h. Cells were washed with PBS three times at 5-min intervals and incubated with secondary antibodies (fluorescein- or Texas Red-conjugated goat anti-rabbit or anti-rat IgG; Jackson Immuno Research, West Grove, PA) at 1:100 with for 1 h. Coverslips were visualized on a Nikon Labphot-2 immunofluorescence microscope or a Bio-Rad 1024 confocal microscope using the 63× oil immersion objective. Images were processed in Adobe Photoshop (Adobe, CA) and figure compiled in Adobe Illustrator (Adobe, CA).

Ca2+ Switch and Measurement of TER-- MDCK cells were plated on 12-mm transwell filter (Costar) at confluence (~3 × 105 cells) and allowed to attach for 24-36 h to form a tight monolayer in normal Ca2+ containing medium (NC). Cells were placed in low Ca2+ (1-4 µM) medium (low calcium) for 1 h followed by switch to NC medium. TER was measured using a Millipore (Bedford, MA) electrical resistance system, and the results are expressed in Omega ·cm2. TER was measured in stable monolayers and during Ca2+ switch in the presence and absence of aluminum fluoride (AlF4-; 3 mm NaF + 50 µM AlCl3, Sigma).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Several lines of evidence have suggested the involvement of heterotrimeric G proteins in tight junction formation. Early studies with G protein modulators such as pertussis toxin, cholera toxin, AlF4-, and a variety of other agents showed variable effects on TJ biogenesis (10). Several confocal studies have localized Galpha i2, Galpha i3, and Galpha 12 in the vicinity of the tight junction (16, 17, 19, 21). Recently, we demonstrated that Galpha o (a member of the Galpha family inhibited by pertussis toxin) expressed in MDCK cells localizes to the subapical lateral membrane overlapping with ZO-1 in the tight junction (16), and this was subsequently confirmed in another study (19). A constitutively active mutant of Galpha o (Q205L) also localizes in this region, and cells expressing Galpha o showed no differences in base-line junctional properties as determined by transepithelial resistance. However, in the Ca2+ switch, MDCK cells expressing activated Galpha o (Q205Lalpha o-MDCK) developed tight junctions at twice the rate and reached significantly higher peak TER values than either Galpha o-MDCK or PC-MDCK cells. Although Galpha o is not normally expressed in epithelia, this observation raises the possibility that one of the Galpha subunits normally found in this location could have a fundamental role in regulating the development and/or maintenance of the TJ.

To further examine the role of G protein alpha  subunits affecting the TJ, we studied the effects of the G protein activator AlF4- on TJ formation in wild type (not transfected; WT-MDCK) and vector (pcDNA3) transfected MDCK cells (PC-MDCK). AlF4- has no known effects on small GTP binding proteins but activates heterotrimeric Galpha subunits. Crystal structures of Galpha i1 obtained with GDP complexed with AlF4- reveal that the position of the gamma -phosphate is occupied by AlF4-. AlF4- in this position prevents catalysis by immobilizing Gln204 and Arg178 (22). Fig. 1 shows that wild type MDCK cells and PC transfected cells cultured on filters develop significantly higher TER in the presence of AlF4-. Untreated steady state TER values were similar between the cell lines, and AlF4- reproducibly increased TER values 3-4-fold. This finding is consistent with activation of one or more endogenous Galpha subunits that results in enhanced steady state resistances.


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Fig. 1.   The effects of aluminum fluoride on base-line TER values in control cells. Untransfected (WT-MDCK) and vector transfected (PC-MDCK) cells were plated at confluence on transwell filters and allowed to stabilize over 48-72 h. Medium was then changed to NC medium (-AlF4-, black bars) or NC medium + (AlF4-)3 (3 mm NaF + 50 µM AlCl3) for 24 h. TER measurements were obtained after 24 h. Results are the means ± S.E. for five experiments each with four to six individual TER values. The differences were significant (p < 0.05) for an effect of AlF4- on both cell lines.

Because AlF4- activates all Galpha subunits in MDCK cells and several studies have placed Galpha i2 in close proximity to the TJ, we tested the hypothesis that Galpha i2 was important to this process by stably expressing Galpha i2 and a constitutively activated Galpha i2 (Q205Lalpha i2) in MDCK cells. The amount of transfected Galpha i2 was determined relative to the levels of endogenous Galpha i2 in PC-MDCK cells. Western blots of the three cell lines using identical amounts of total protein were analyzed (not shown) using NIH image (Wayne Rasband, NIH). Relative to PC-MDCK cells, the level of Galpha i2 in Galpha i2-MDCK cells was 3.9 ± 0.4-fold (n = 7) increased, and for Q205Lalpha i2-MDCK the level was of Galpha i2 was 1.8 ± 0.2-fold (n = 7) above PC-MDCK cells. To confirm that constitutively activated Galpha i2 was expressed in these transfected MDCK cells, we utilized a tryptic cleavage analysis of Galpha i2 (Fig. 2). This technique has been widely utilized as an indicator of Galpha subunit conformation (23, 24) and is based on the observation that Galpha subunits have a different cleavage pattern depending on whether they are folded into an active or inactive conformation. In the active conformation (GTP-liganded), there is only a single tryptic site accessible near the N terminus (approximately Arg21) resulting in a slightly truncated protein (39 kDa instead of 41 kDa for Galpha i2). In the inactive (GDP-liganded) conformation, an additional site becomes accessible in the alpha 2 helix or switch region (near Arg209) resulting in peptides of approximately 25 and 17 kDa. Fig. 2 demonstrates the tryptic cleavage patterns of cell homogenates from each of the transfected cell lines (PC-, Galpha i2-, and Q205Lalpha i2-MDCK cells). In PC- and Galpha i2-transfected cells, untreated Galpha i2 migrates at 41 kDa (first lane of each set) and is stabilized in the active conformation (39 kDa) if preincubated with the nonhydrolyzable GTP analogue, GTPgamma S (last lane of each set). However, in the absence of added nucleotide (middle lane of each set) the Galpha subunits should be GDP bound from endogenous GTP/GDP in the cell, and Galpha i2 is cleaved into 25- and 17-kDa fragments with no detectable 39-kDa peptide. The 25-kDa fragment (derived from the N terminus) is not detectable with the AS7 antibody, and the 17-kDa fragment is more labile (25) and consequently is not well visualized on these blots (not shown). Tryptic digestion of Q205Lalpha i2-transfected cells shows that in the absence of added nucleotide (middle lane), there is a fraction of Galpha i2 that is tryptic-resistant, indicating persistence of Galpha i2 in an "active" conformation. The amount of Galpha i2 in the active conformation of Q205Lalpha i2-MDCK cells is a small fraction of the starting material. This is due, in part, to the observation that Galpha subunits with mutations that result in an active conformation are more sensitive to proteolysis than wild type Galpha subunits activated with GTPgamma S (20). In addition, it is necessary to do these studies on whole cell lysates in the absence of protease inhibitors. The Western blots were deliberately overexposed to look for bands migrating at 39 kDa. This analysis confirms expression of constitutively activated Galpha i2 in Q205Lalpha i2-MDCK cells.


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Fig. 2.   Tryptic proteolysis of PC-, Galpha i2-, and Q205Lalpha i2-MDCK cells. Confluent monolayers of each cell type were washed with PBS, scraped, and fractionated as described under "Experimental Procedures." Approximately 50-75 µg of total protein was used; samples were preincubated at 30 °C for 10 min with no added nucleotide (-) or 100 µM GTPgamma S (+) followed by digestion with 20 pmol of trypsin for 20 min at 30 °C. Reactions were terminated with SDS-polyacrylamide gel electrophoresis sample buffer and boiling. Samples were analyzed by Western with AS7 antibody at 1:1000 and ECL as described under "Experimental Procedures." The times of exposure for each cell line differ and were deliberately overexposed to determine whether the expected 39-kDa peptide could be detected. Only the Q205Lalpha i2-MDCK cells demonstrated a protected fragment at 39 kDa in the absence of added nucleotide.

We have previously demonstrated that WT-MDCK cells express some Galpha i2 in the subapical lateral membrane overlapping with ZO-1 (16). To eliminate the possibility that the transfection process affects Galpha i2 localization, PC-MDCK cells were characterized by confocal microscopy. Fig. 3A shows a confocal image of PC-MDCK cells costained with antibodies to ZO-1 and Galpha i2 (used at 1:25 dilution). The confocal images reveal that Galpha i2 partially colocalizes with ZO-1 at the level of the tight junction. There is significant intracellular staining that was also seen in WT-MDCK cells (16). To determine whether transfected Galpha i2 subunits were localized in a similar manner to the endogenous Galpha i2, the Galpha i2 antibody (AS7) was diluted to a point where the endogenous Galpha i2 was barely detectable. Fig. 3B shows a confocal analysis of PC-, Galpha i2-, and Q205Lalpha i2-MDCK stained and analyzed under identical conditions using a 1:100 dilution of the Galpha i2 antibody. In panel a, PC-MDCK cells only demonstrate faint intracellular staining, but in panels b and c, transfected Galpha i2 and Q205Lalpha i2 can be visualized in the subapical lateral membrane overlapping with the TJ marker, ZO-1. Again, there is intracellular staining that is similar to the endogenous Galpha i2 (Fig. 3A). Overall the pattern of transfected Galpha i2 and Q205Lalpha i2 is very similar to that seen with the endogenous Galpha i2 subunits. These results confirm that transfected Galpha i2 and Q205Lalpha i2 partition between the lateral membrane overlapping with the TJ and intracellular compartments. This finding is similar to our prior findings with Galpha o-transfected MDCK cells (16).


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Fig. 3.   Confocal localization of Galpha i2 in transfected cells. A, PC-MDCK cells were grown on Transwell filters and double stained with antibody to Galpha i2 (AS7) at 1:25 dilution and undiluted ZO-1 monoclonal antibody as described under "Experimental Procedures." A single field was simultaneously visualized for ZO-1 and Galpha i2 localization. Magnification, 950×. B, transfected cells were simultaneously stained under identical conditions using AS7 antibody at 1:100 dilution and undiluted ZO-1 as described under "Experimental Procedures": PC-MDCK cells (panel a), Galpha i2-MDCK (panel b), and Q205Lalpha i2-MDCK (panel c). Identical fields were simultaneously visualized by confocal microscopy as described. Magnification, 950×.

Because transfected Galpha i2 and Q205Lalpha i2 were localized in a manner similar to that of the endogenous Galpha i2, we next determined whether Galpha i2 localization in the TJ had any functional consequences for the tight junction. PC-, Galpha i2-, and Q205Lalpha i2-MDCK cells were simultaneously analyzed under steady state conditions and also by using the Ca2+ switch. TJ integrity was followed by measurement of transepithelial resistance. Fig. 4A demonstrates the base-line resistances in these cells and the pattern of TER development after the Ca2+ switch. Overexpressing Galpha i2 had a small but insignificant (p = 0.07) effect on base-line resistances in comparison with PC-MDCK cells (576 ± 89 versus 377 ± 52; n = 12), but Q205Lalpha i2-MDCK cells had significantly higher base-line TER values (1187 ± 150 Omega ·cm2; p < 0.001). The base-line TER values for Galpha i2- and PC-MDCK cells were similar to reported values of Galpha o (16), and several clones were analyzed with no significant differences seen among the clones. To gain insight into the mechanism of higher TER values observed in Q205Lalpha i2-MDCK cells, all three cell lines were simultaneously analyzed in the Ca2+ switch. The elevated TER in Q205Lalpha i2-MDCK cells could be achieved by differences in the kinetics of TER development. Nonlinear regression analysis of the TER data between 0-12 h for all of the cell lines (Fig. 4A) indicates an asymptotic approach to peak TER. Although the data do not precisely fit standard kinetic models, the kinetics of TER development in these cells is similar to what has been reported in other studies (15, 26). The time to half-maximal TER is a useful value for discussing the effects of Galpha i2 expression on TER biogenesis, and these values were calculated for each cell line in the presence and absence of AlF4- (Table I). Table I shows that the time to half-maximal TER (T50) was significantly more rapid in Galpha i2- and Q205Lalpha i2-MDCK cells (0.8 ± 0.3 and 1.2 ± 0.3 h, respectively) in comparison with PC cells (3.0 ± 0.5 h). AlF4- had no significant on Galpha i2-transfected cells but significantly shortened T50 for PC cells (1.1 ± 0.7 h, a value similar to that of Galpha i2 cells). The observation that PC-MDCK cells treated with AlF4- develop TER nearly as rapidly as Q205Lalpha i2-MDCK cells suggests that Galpha i2 may be the predominant Galpha subunit critical in TER development. Fig. 4B shows that the base-line TER values for the three cell lines on the day following the Ca2+ switch (26 h) are significantly higher in the presence of ALF4-. In contrast to AlF4- effects on the rate of TER development, all three cell lines had significantly increased base-line TER at 26 h in the presence of AlF4-. Similar effects of AlF4- were seen with the three cell lines cultured in the steady state (not shown). This raises the possibility that AlF4- activates additional Galpha subunits in the steady state that enhances transepithelial resistance.


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Fig. 4.   Base-line TER values and time course of TER development after Ca2+ switch. PC-, Galpha i2-, and Q205Lalpha i2-MDCK cells were plated at confluent density on Transwell filters, and base-line TER values were obtained 36-40 h later. Cells were then placed in low calcium medium and TJ biogenesis followed over time using the Ca2+ switch as described under "Experimental Procedures." Blanks were subtracted for each experiment. A, base line (BL) and time course of TER development. TER values were significantly higher for Q205Lalpha i2-MDCK cells (1187 ± 150 Omega ·cm2; p < 0.001) than for Galpha i2-MDCK (576 ± 89 Omega ·cm2) or PC-MDCK (377 ± 52 Omega ·cm2). The difference between Galpha i2- and PC-MDCK was not significantly different. TER values were obtained every 2 h after switching from low calcium to NC medium at time 0. Post-Ca2+ switch base-line values are shown at 26 h. There was a decrease in base-line TER values for each of the three cell lines, but Q205Lalpha i2 remained significantly higher than the other two cell types. Results are expressed as the means ± S.E. of 12 independent experiments with n = 4-6 for each cell line in each experiment. Graphs were generated and statistical analyses were performed on data using Graphpad Prism 2.0 (Graphpad Software, Inc.) B, post-Ca2+ switch base-line TER values obtained at 26 h for the six independent experiments ± AlF4-. The differences ± AlF4- were significant for each of the cell lines (p = 0.008 for PC and p = 0.002 for both Galpha i2 and Q205Lalpha i2-MDCK cells).

                              
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Table I
Summary of times to half-maximal TER for Galpha i2-, Q205Lalpha i2-, and PC-MDCK cells
Ca+2 switch TER data from 0-12 h for each cell line was analyzed by nonlinear regression using GraphPad Prism (San Diego, CA). TER data was fit to a hyperbola
<FENCE><FR><NU><UP>TER Max</UP>×T</NU><DE>T<SUB>50</SUB>+T</DE></FR>=Y</FENCE>
for each transfected cell line in the absence (n = 12) and presence of AlF4- (n = 6; added in the low calcium medium and continued after the switch). TER Max is the line defined by the maximal TER obtained between 6-12 h and was held constant for calculation of the time to one-half maximal TER (T50). The differences in T50 between PC-MDCK cells and Galpha i2- and Q205Lalpha i2-MDCK cells were significant (p < 0.02), and the difference with or without AlF4- was significant only for PC cells (p < 0.05).

Taken together, these studies offer direct evidence that Galpha i2 is a critical regulator of tight junction biogenesis and affects base-line characteristics of the tight junction. The protein composition of the TJ is complex with one integral membrane protein identified so far (occludin), several peripherally attached proteins with partially defined functions (including ZO-1, -2, and -3) and a variety of signal transduction molecules including PKC isoforms, Galpha subunits, and tyrosine kinases (see Ref. 5 for review). How these diverse proteins function to maintain and regulate the development of tight junctions is not well understood. G proteins could be activated within the TJ through a classical seven-transmembrane receptor (although none yet identified in the TJ) or alternatively through a modulatory protein that promotes GDP release or slows GTP hydrolysis. Additional transmembrane proteins must exist within the TJ (27), and there are multiple examples of modulatory proteins that affect G protein function. GTPase activating proteins (RGS proteins; regulators of G protein signaling; reviewed in Ref. 28) interact with Galpha subunits, and nucleotide exchange factors that promote GDP release have been described for many small G proteins such as Ras (29). Although analogous proteins for Galpha subunits have not yet been identified, such proteins may exist and could provide mechanisms for activation of Galpha subunits in the TJ or within intracellular compartments (30). Our findings that Galpha i2 is important for both the maintenance and development of the TJ does not exclude roles for other Galpha subunits, and in fact the effects of AlF4- on the steady state TER suggests that other Galpha subunits are likely to enhance this barrier.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM55223 (to B. M. D.) and DK53507 (to S. K. N.) and a March of Dimes Basil O'Connor Award (to B. M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a National Research Service Award.

§ Established Investigator of the American Heart Association.

To whom correspondence should be addressed: Harvard Inst. of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker{at}rics.bwh.harvard.edu.

The abbreviations used are: TJ, tight junction; MDCK, Madin-Darby canine kidney; G protein, guanine nucleotide-binding protein; ZO, zona occludens; TER, transepithelial resistance; PKC, protein kinase C; NC, normal calcium; PBS, phosphate-buffered saline; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., and Tsukita, S. (1993) J. Cell Biol. 123, 1777-1788[Abstract]
  2. Stevenson, B. R., and Goodenough, D. A. (1984) J. Cell Biol. 98, 1209-1221[Abstract]
  3. Stevenson, B. R., Silicano, J. D., Mooseker, M. S., and Goodenough, D. A. (1986) J. Cell Biol. 103, 755-766[Abstract]
  4. Haskins, J., Gu, L., Wittchen, E. S., Hibbard, J., and Stevenson, B. R. (1998) J. Cell Biol. 141, 199-208[Abstract/Free Full Text]
  5. Denker, B. M., and Nigam, S. K. (1998) Am. J. Physiol. 274, F1-F9[Medline] [Order article via Infotrieve]
  6. Rodriguez-Boulan, E., and Nelson, W. J. (1989) Science 245, 718-725[Medline] [Order article via Infotrieve]
  7. Stuart, R. O., Sun, A., Bush, K. T., and Nigam, S. K. (1996) J. Biol. Chem. 271, 13636-13641[Abstract/Free Full Text]
  8. Stuart, R. O., and Nigam, S. K. (1995) Semin. Nephrol. 15, 315-326[Medline] [Order article via Infotrieve]
  9. Nigam, S. K., Rodriguez-Boulan, E., and Silver, R. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6162-6166[Abstract]
  10. Balda, M. S., Gonzalez-Mariscal, L., Macias-Silva, M., Torres-Marquez, M. E., Garcia Sainz, J. A., and Cereijido, M. (1991) J. Membr. Biol. 122, 193-202[Medline] [Order article via Infotrieve]
  11. Cereijido, M., Gonzalez-Mariscal, L., Conreras, R. G., Gallardo, J. M., Garcia- Villegas, R., and Valdes, J. (1993) J. Cell Sci. 17, 127-132
  12. Gumbiner, B. M. (1996) Cell 84, 345-357[Medline] [Order article via Infotrieve]
  13. Stuart, R. O., Sun, A., Panichas, M., Hebert, S. C., Brenner, B. M., and Nigam, S. K. (1994) J. Cell. Physiol. 159, 423-433[Medline] [Order article via Infotrieve]
  14. Rosson, D., O'Brien, T. G., Kampherstein, J. A., Szallasi, Z., Bogi, K., Blumberg, P. M., and Mullin, J. M. (1997) J. Biol. Chem. 272, 14950-14953[Abstract/Free Full Text]
  15. Stuart, R. O., and Nigam, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6072-6076[Abstract/Free Full Text]
  16. Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753[Abstract/Free Full Text]
  17. de Almeida, J. B., Holtzman, E. J., Peters, P., Ercolani, L., Ausiello, D. A., and Stow, J. L. (1994) J. Cell Sci. 107, 507-515[Abstract/Free Full Text]
  18. Senkfor, S. I., Johnson, G. L., and Berl, T. (1993) J. Clin. Invest. 92, 786-790[Medline] [Order article via Infotrieve]
  19. Hamilton, S. E., and Nathanson, N. M. (1997) Biochem. Biophys. Res. Commun. 234, 1-7[CrossRef][Medline] [Order article via Infotrieve]
  20. Denker, B. M., Boutin, P. M., and Neer, E. J. (1995) Biochemistry 34, 5544-5553[Medline] [Order article via Infotrieve]
  21. Dodane, V., and Kachar, B. (1996) J. Membr. Biol. 149, 199-209[CrossRef][Medline] [Order article via Infotrieve]
  22. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412[Medline] [Order article via Infotrieve]
  23. Denker, B. M., Schmidt, C. J., and Neer, E. J. (1992) J. Biol. Chem. 267, 9998-10002[Abstract/Free Full Text]
  24. Denker, B. M., Neer, E. J., and Schmidt, C. J. (1992) J. Biol. Chem. 267, 6272-6277[Abstract/Free Full Text]
  25. Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229[Abstract/Free Full Text]
  26. Balda, M. S., Gonzalez-Mariscal, L., Matter, K., Cereijido, M., and Anderson, J. M. (1993) J. Cell Biol. 123, 293-302[Abstract]
  27. Saitou, M., Fujimoto, K., Doi, Y., Itoh, M., Fujimoto, T., Furuse, M., Takano, H., Noda, T., and Tsukita, S. (1998) J. Cell Biol. 141, 397-408[Abstract/Free Full Text]
  28. Berman, D. M., and Gilman, A. G. (1998) J. Biol. Chem. 273, 1269-1272[Free Full Text]
  29. Shou, C., Farnsworth, C. L., Neel, B. G., and Feig, L. A. (1992) Nature 358, 351-354[CrossRef][Medline] [Order article via Infotrieve]
  30. Denker, S. P., McCaffery, J. M., Palade, G. E., Insel, P. A., and Farquhar, M. G. (1996) J. Cell Biol. 133, 1027-1040[Abstract]


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