Involvement of Gαi2 in the Maintenance and Biogenesis of Epithelial Cell Tight Junctions

Abstract

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 α subunits) results in a 3–4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750–25753). Gαi2 has been localized within the tight junction, and a role for Gαi2in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gαi2, or a GTPase-deficient mutant (constitutively activated), Q205Lαi2. Tryptic conformational analysis confirmed expression of a constitutively active Gαi2 in Q205Lαi2-MDCK cells, and confocal microscopy showed a similar pattern of Gαi2 localization in the three cell lines. Q205Lαi2-MDCK cells had significantly higher base-line TER values than wild type Gαi2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gαi2); 377 ± 52 Ω·cm2 (PC)), and both Gαi2- and Q205Lαi2-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 Gαi2- or Q205Lαi2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4− (similar times to Gαi2-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 Gαi2 is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role. 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 α subunits) results in a 3–4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750–25753). Gαi2 has been localized within the tight junction, and a role for Gαi2in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gαi2, or a GTPase-deficient mutant (constitutively activated), Q205Lαi2. Tryptic conformational analysis confirmed expression of a constitutively active Gαi2 in Q205Lαi2-MDCK cells, and confocal microscopy showed a similar pattern of Gαi2 localization in the three cell lines. Q205Lαi2-MDCK cells had significantly higher base-line TER values than wild type Gαi2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gαi2); 377 ± 52 Ω·cm2 (PC)), and both Gαi2- and Q205Lαi2-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 Gαi2- or Q205Lαi2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4− (similar times to Gαi2-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 Gαi2 is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role. 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) 1The abbreviations used are: TJtight junctionMDCKMadin-Darby canine kidneyG proteinguanine nucleotide-binding proteinZOzona occludensTERtransepithelial resistancePKCprotein kinase CNCnormal calciumPBSphosphate-buffered salineGTPγSguanosine 5′-3-O-(thio)triphosphate. 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 (1Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar). There are several peripherally attached membrane proteins found in the TJ including the zona occludens family (ZO-1, -2, and -3) (2Stevenson B.R. Goodenough D.A. J. Cell Biol. 1984; 98: 1209-1221Crossref PubMed Scopus (99) Google Scholar, 3Stevenson B.R. Silicano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1289) Google Scholar, 4Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (495) Google Scholar). ZO proteins are members of the MAGUK (membrane associatedguanylate 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. 5Denker B.M. Nigam S.K. Am. J. Physiol. 1998; 274: F1-F9Crossref PubMed Google Scholar). tight junction Madin-Darby canine kidney guanine nucleotide-binding protein zona occludens transepithelial resistance protein kinase C normal calcium phosphate-buffered saline guanosine 5′-3-O-(thio)triphosphate. 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 (6Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (819) Google Scholar). The Ca2+ switch model of TJ formation in MDCK cells has been widely utilized to gain insights into the function of polarized epithelial cells (7Stuart R.O. Sun A. Bush K.T. Nigam S.K. J. Biol. Chem. 1996; 271: 13636-13641Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 8Stuart R.O. Nigam S.K. Semin. Nephrol. 1995; 15: 315-326PubMed Google Scholar, 9Nigam S.K. Rodriguez-Boulan E. Silver R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6162-6166Crossref PubMed Scopus (73) Google Scholar, 10Balda M.S. Gonzalez-Mariscal L. Macias-Silva M. Torres-Marquez M.E. Garcia Sainz J.A. Cereijido M. J. Membr. Biol. 1991; 122: 193-202Crossref PubMed Scopus (247) Google Scholar, 11Cereijido M. Gonzalez-Mariscal L. Conreras R.G. Gallardo J.M. Garcia- Villegas R. Valdes J. J. Cell Sci. 1993; 17: 127-132Crossref Google Scholar) and recapitulates many of the critical molecular events of epithelial morphogenesis. MDCK cells cultured in low calcium (μm) 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 (12Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar). 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 (9Nigam S.K. Rodriguez-Boulan E. Silver R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6162-6166Crossref PubMed Scopus (73) Google Scholar), and chelation of intracellular calcium perturbs TER development (13Stuart R.O. Sun A. Panichas M. Hebert S.C. Brenner B.M. Nigam S.K. J. Cell. Physiol. 1994; 159: 423-433Crossref PubMed Scopus (105) Google Scholar). 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 (7Stuart R.O. Sun A. Bush K.T. Nigam S.K. J. Biol. Chem. 1996; 271: 13636-13641Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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. PKCζ and PKCα, have also been localized in the vicinity of the TJ (14Rosson D. O'Brien T.G. Kampherstein J.A. Szallasi Z. Bogi K. Blumberg P.M. Mullin J.M. J. Biol. Chem. 1997; 272: 14950-14953Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 15Stuart R.O. Nigam S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6072-6076Crossref PubMed Scopus (245) Google Scholar, 16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar). We previously demonstrated that expressing a constitutively activated Gαo (Q205L) in MDCK cells significantly accelerated TJ biogenesis without affecting base-line resistances. Although Gαo is a member of the G protein family inhibited by pertussis toxin (∼80% similar to Gαi1–3), its receptors and effectors are distinct, and furthermore, Gαo is not detected in renal epithelia or MDCK cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Senkfor S.I. Johnson G.L. Berl T. J. Clin. Invest. 1993; 92: 786-790Crossref PubMed Scopus (9) Google Scholar, 19Hamilton S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google Scholar). Several Gαi family members are expressed in epithelial cells, and Gαi2 has been shown to overlap with the tight junction in epithelial cell lines (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar). Taken together, these observations raise the possibility that Gαi2 may be an important regulator of tight junctions. To test this hypothesis, we initially looked for effects of AlF4− (activator of Gα subunits) on tight junctions in control cell lines and then established MDCK cell lines overexpressing wild type Gαi2 and a constitutively activated Gαi2 (GTPase-deficient, Q205Lαi2). 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 Gαi2, indicating that this Gα subunit is critical to the development and maintenance of tight junctions. Rat Gαi2 cDNA was cloned into Bluescript (Stratagene) as described previously (20Denker B.M. Boutin P.M. Neer E.J. Biochemistry. 1995; 34: 5544-5553Crossref PubMed Scopus (37) Google Scholar) and recloned into the EcoRI andApaI sites of pcDNA3 (Invitrogen). Q205Lαi2 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.) Subconfluent MDCK cells (ATCC, Manassas, VA) were transfected with 10 μg of linearized plasmid by calcium phosphate precipitation method as described previously (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). G418-resistant colonies were analyzed for increased Gαi2expression by Western blot using a rabbit polyclonal antibody directed toward the C terminus of Gαi2 (AS7; NEN Life Science Products). Control cells were obtained by transfecting pcDNA3 without insert, and all cell lines were established in parallel. Confluent or Q205Lαi2 cells were with and then into were and three times and times a were at with or were on and was were at for and was by the of followed by for were then analyzed by and Western blot using rabbit polyclonal antibody and with or Q205Lαi2-MDCK cells were on or with and with for 10 were then with and as described previously (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). were with rabbit polyclonal Gαi2 from NEN Life Science at several and to ZO-1 of D. for were with three times at and with or or at with for were on a or a confocal using the were in and in MDCK cells were on at and to for to a tight in normal Ca2+ medium were in low Ca2+ medium for followed by switch to TER was using a resistance and the results are expressed in TER was in monolayers and during Ca2+ switch in the presence and of Several lines of have the of heterotrimeric G proteins in tight junction studies with G protein as pertussis and a variety of showed effects on TJ biogenesis M.S. Gonzalez-Mariscal L. Macias-Silva M. Torres-Marquez M.E. Garcia Sainz J.A. Cereijido M. J. Membr. Biol. 1991; 122: 193-202Crossref PubMed Scopus (247) Google Scholar). Several confocal studies have localized Gαi2, and in the vicinity of the tight junction (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar, 19Hamilton S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google B. J. Membr. Biol. 1996; PubMed Scopus Google Scholar). we demonstrated that Gαo member of the Gα family inhibited by pertussis expressed in MDCK cells to the membrane with ZO-1 in the tight junction (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and this was confirmed in study S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google Scholar). constitutively active mutant of Gαo (Q205L) also in this and cells expressing in base-line junctional as by transepithelial in the Ca2+ switch, MDCK cells expressing activated developed tight junctions at the and reached significantly higher TER values than or PC-MDCK Although Gαo is not expressed in this the possibility that of the Gα subunits found in this have a role in regulating the development and/or maintenance of the TJ. To the role of G protein α subunits affecting the TJ, we studied the effects of the G protein AlF4− on TJ formation in wild type and vector transfected MDCK cells AlF4− has effects on proteins but heterotrimeric Gα of obtained with with AlF4− that the of the is by AlF4−. AlF4− in this prevents by and E. M.E. Science. 1994; PubMed Scopus Google Scholar). that wild type MDCK cells and PC transfected cells cultured on develop significantly higher TER in the presence of AlF4−. TER values were similar the cell and AlF4− increased TER values is with of or more Gα subunits that results in resistances. Because AlF4− all Gα subunits in MDCK cells and several studies have Gαi2 in proximity to the TJ, we the that Gαi2 was important to this by expressing Gαi2 and a constitutively activated Gαi2 in MDCK The of transfected Gαi2 was to the of Gαi2 in PC-MDCK Western of the three cell lines using of protein were analyzed using to PC-MDCK cells, the of Gαi2 in cells was ± and for Q205Lαi2-MDCK the was of Gαi2 was ± PC-MDCK To that constitutively activated Gαi2 was expressed in these transfected MDCK cells, we utilized a analysis of Gαi2 has been widely utilized as an indicator of Gα subunit B.M. Neer E.J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, B.M. Neer E.J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) and is on the that Gα subunits have a different pattern on they are into an active or In the active is only a near the terminus in a protein of for In the an additional in the or switch in of and the of cell from of the transfected cell lines and Q205Lαi2-MDCK cells). In and Gαi2-transfected cells, at of and is in the active with the of in the of of the Gα subunits be from in the and Gαi2 is into and with The from the is not with the and the is more E.J. J.M. J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar) and is not well on these Tryptic of Q205Lαi2-transfected cells that in the of is a of Gαi2 that is indicating of Gαi2 in an The of Gαi2 in the active of Q205Lαi2-MDCK cells is a of the is in to the that Gα subunits with that in an active are more to than wild type Gα subunits activated with (20Denker B.M. Boutin P.M. Neer E.J. Biochemistry. 1995; 34: 5544-5553Crossref PubMed Scopus (37) Google Scholar). In is to these studies on cell in the of The Western were to for at analysis expression of constitutively activated Gαi2 in Q205Lαi2-MDCK We have previously demonstrated that cells Gαi2 in the membrane with ZO-1 (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To the possibility that the transfection Gαi2 PC-MDCK cells were by confocal a confocal of PC-MDCK cells with to ZO-1 and at The confocal that Gαi2 partially with ZO-1 at the of the tight There is intracellular that was also in cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To transfected Gαi2 subunits were localized in a similar to the Gαi2, the Gαi2 antibody was to a the Gαi2 was a confocal analysis of and Q205Lαi2-MDCK and analyzed using a of the Gαi2 In PC-MDCK cells only intracellular but in transfected Gαi2 and Q205Lαi2 can be in the membrane with the TJ is intracellular that is similar to the Gαi2 the pattern of transfected Gαi2 and Q205Lαi2 is similar to that with the Gαi2 These results that transfected Gαi2 and Q205Lαi2 the membrane with the TJ and intracellular is similar to prior findings with MDCK cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Because transfected Gαi2 and Q205Lαi2 were localized in a similar to that of the Gαi2, we in the TJ had for the tight and Q205Lαi2-MDCK cells were analyzed and also by using the Ca2+ switch. TJ integrity was followed by of transepithelial the base-line resistances in these cells and the pattern of TER development after the Ca2+ switch. Gαi2 had a but effect on base-line resistances in with PC-MDCK cells ± 377 ± but Q205Lαi2-MDCK cells had significantly higher base-line TER values (1187 ± 150 The base-line TER values for Gαi2- and PC-MDCK cells were similar to values of Gαo (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and several were analyzed with the To gain into the of higher TER values in Q205Lαi2-MDCK cells, all three cell lines were analyzed in the Ca2+ switch. The TER in Q205Lαi2-MDCK cells be by in the kinetics of TER analysis of the TER for all of the cell lines an to Although the not models, the kinetics of TER development in these cells is similar to has been in studies R.O. Nigam S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6072-6076Crossref PubMed Scopus (245) Google Scholar, M.S. Gonzalez-Mariscal L. K. Cereijido M. J.M. J. Cell Biol. 1993; 123: PubMed Scopus Google Scholar). The to half-maximal TER is a for the effects of Gαi2expression on TER biogenesis, and these values were for cell line in the presence and of AlF4− that the to half-maximal TER was significantly more rapid in Gαi2- and Q205Lαi2-MDCK cells ± and ± in with PC cells ± AlF4− had on Gαi2-transfected cells but significantly for PC cells ± a similar to that of Gαi2 cells). The that PC-MDCK cells with TER as rapidly as Q205Lαi2-MDCK cells suggests that Gαi2 may be the Gα subunit critical in TER that the base-line TER values for the three cell lines on the the Ca2+ switch are significantly higher in the presence of In to AlF4− effects on the of TER all three cell lines had significantly increased base-line TER at in the presence of AlF4−. effects of AlF4− were with the three cell lines cultured in the the possibility that AlF4− additional Gα subunits in the that transepithelial of times to half-maximal TER for and PC-MDCK ± ± ± ± ± ± switch TER from for cell line was analyzed by using TER was to a TER transfected cell line in the and presence of in the low calcium medium and after the TER is the line by the TER obtained and was for of the to TER The in PC-MDCK cells and Gαi2- and Q205Lαi2-MDCK cells were and the with or without AlF4− was only for PC cells in a switch TER from for cell line was analyzed by using TER was to a TER transfected cell line in the and presence of in the low calcium medium and after the TER is the line by the TER obtained and was for of the to TER The in PC-MDCK cells and Gαi2- and Q205Lαi2-MDCK cells were and the with or without AlF4− was only for PC cells Taken together, these studies that Gαi2 is a critical regulator of tight junction biogenesis and base-line characteristics of the tight The protein of the TJ is complex with membrane protein identified so far several peripherally attached proteins with partially functions -2, and -3) and a variety of signal transduction molecules including PKC Gα and Ref. 5Denker B.M. Nigam S.K. Am. J. Physiol. 1998; 274: F1-F9Crossref PubMed Google for these proteins function to maintain and the development of tight junctions is not well G proteins be activated within the TJ a identified in the or a protein that or transmembrane proteins within the TJ M. K. Itoh M. T. M. T. Tsukita S. J. Cell Biol. 1998; 141: PubMed Scopus Google Scholar), and are of proteins that G protein proteins of G protein in Ref. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) with Gα and that promote have been described for many G proteins as C. 1992; PubMed Scopus Google Scholar). Although proteins for Gα subunits have not been proteins may and mechanisms for of Gα subunits in the TJ or within intracellular J.M. J. Cell Biol. 1996; PubMed Scopus Google Scholar). findings that Gαi2 is important for both the maintenance and development of the TJ not roles for Gα and in the effects of AlF4− on the TER suggests that Gα subunits are likely to this