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J. Biol. Chem., Vol. 277, Issue 28, 24855-24858, July 12, 2002

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ACCELERATED PUBLICATION
Zonula Occludens-1 Is a Scaffolding Protein for Signaling Molecules

G₁₂ DIRECTLY BINDS TO THE Src HOMOLOGY 3 DOMAIN AND REGULATES PARACELLULAR PERMEABILITY IN EPITHELIAL CELLS^*

Tobias N. Meyer^§, Catherine Schwesinger^¶, and Bradley M. Denker^**

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

Received for publication, April 18, 2002, and in revised form, May 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Zonula occludens proteins are multidomain proteins usually localized at sites of intercellular junctions, yet little is known about their role in regulating junctional properties. Multiple signaling proteins regulate the junctional complex, and several (including G proteins) have been co-localized with zonula occludens-1 (ZO-1) in the tight junction of epithelial cells. However, evidence for direct interactions between signaling proteins and tight junction proteins has been lacking. In these studies, we constructed G-glutathione S-transferase (GST) fusion proteins and tested for interactions with [³⁵S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only G₁₂ directly interacted with in vitro translated ZO-1 and ZO-2. Using a series of ZO-1 domains expressed as GST fusion proteins and in vitro translated [³⁵S]methionine-labeled G₁₂, we found that G₁₂ and constitutively active (Q229L) ₁₂ (QL₁₂) bind to the Src homology 3 (SH3) domain of ZO-1. This binding was not detected with SH3 domains from other proteins. Inducible expression of wild-type ₁₂ and QL₁₂ in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In G₁₂-expressing cells, we found that ZO-1 and G₁₂ co-localize by confocal microscopy and co-immunoprecipitate. G₁₂ from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QL₁₂ in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with G₁₂ and that G₁₂ regulates barrier function of MDCK cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To provide a barrier function, epithelial cells have evolved a highly organized junctional complex that includes tight junctions (TJs),¹ adherens junctions, gap junctions, and desmosomes. A variety of proteins have been identified within the junctional complex; they include integral membrane proteins (such as claudins, occludin, and E-cadherin), signaling molecules (protein kinase C, Src tyrosine kinases, small G proteins, and heterotrimeric G subunits), and potential scaffolding proteins (ZO-1, ZO-2, and ZO-3; for reviews, see Refs. 1 and 2). The ZO proteins are multidomain MAGUK (membrane-associated guanylate kinase) family members that contain a potential guanylate kinase domain, multiple PDZ domains, and a SH3 domain (see Fig. 1). Although ZO-1 is expressed in multiple cell types, in epithelial cells it is localized only in the tight junction, the most apical component of the junctional complex. The two related family members, ZO-2 and ZO-3, form a complex with ZO-1 in the TJ and link to the actin cytoskeleton (3). The function of ZO proteins in regulating the junction is unknown, but based upon their domain structure, they have been proposed to be scaffolding proteins (4).

Several G subunits, including G₁₂, partially co-localize within the epithelial cell junction (1). Regulation of the junctional complex by heterotrimeric G proteins was suggested in early experiments (5). Subsequent studies have demonstrated that pertussis toxin-sensitive family members, G_i2 and G_o, localize in the tight junction of Madin-Darby canine kidney (MDCK) cells and affect both tight junction assembly and base-line properties (6, 7). Recently we showed that G_s stimulates tight junction assembly and co-localizes with a ZO-1 complex (8). However, to date, there has been no direct demonstration of an interaction between G protein signaling molecules and TJ proteins. Utilizing in vitro binding studies and inducible expression in MDCK cells, we demonstrate that G₁₂ directly binds to ZO-1 through the SH3 domain and that activated G₁₂ regulates paracellular permeability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Reagents-- cDNAs for mouse wild-type ₁₂ (wt₁₂) and constitutively active ₁₂ (Q229L) QL₁₂ were provided by Henry Bourne (University of California, San Francisco), G₁₃ was provided by D. Barber (University of California, San Francisco), and ZO-1, ZO-2, and anti-ZO-1 rat monoclonal antibody (R40.76) were from Dr. Goodenough (Harvard Medical School, Boston, MA). ZO-1-GST fusion protein constructs were the generous gift of M. Balda (University College, London) and are described in Ref. 9. The SH3 domains of n-Src, c-Src, Crk, Abl, and Grb2 were provided by Bruce Mayer (Children's Hospital, Boston, MA) (10). G₁₂ antibodies were from Santa Cruz Biotechnology (S-20, Santa Cruz, CA). Tet-Off MDCK type II epithelial cells (MDCK-T23), Tet-Off cloning vectors, and Tet-free fetal calf serum were obtained from CLONTECH (Palo Alto, CA).

In Vitro Protein Binding Studies-- G_i2-, G_s-, G_q-, G₁₂-, and G₁₃-GST fusion protein constructs were cloned as N-terminal GST-G fusion proteins using standard techniques into pGEX4T (Amersham Biosciences). GST fusion proteins were expressed in Escherichia coli and purified from bacterial lysates as described previously (11). In vitro translation of the G subunits, ZO-1, and ZO-2 was performed using [³⁵S]methionine in a coupled rabbit reticulocyte lysate system (Promega, Madison, WI) as described previously (11). Similar amounts of ³⁵S-labeled proteins were incubated with 1 µg of GST fusion protein prebound to glutathione-agarose 4B overnight at 4 °C in 50 mM Tris-HCl, pH 7.4, 75 mM sucrose, 6 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 (buffer A). Samples were washed in buffer A, eluted, and analyzed by SDS-PAGE and autoradiography.

Tet-Off Inducible MDCK Cell Lines-- wt₁₂ and QL₁₂ were subcloned into the pTRE expression vector (CLONTECH) by standard techniques. Tet-Off MDCK cells were cultured as described by Jou et al. (12). Cells were transfected using linearized plasmid and pTK-Hyg with FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. Resistant clones were maintained in medium containing 100 µg/ml hygromycin, 100 µg/ml G418, and 40 ng/ml doxycycline. Subsequent experiments were carried out in medium without hygromycin and G418 and with or without doxycycline.

Immunohistochemistry-- G₁₂- and QL₁₂-transfected Tet-Off MDCKcells were grown to confluency with or without doxycycline for specific times. Cells were fixed with ice cold 100% methanol for 20 min at 20 °C and blocked for 45 min at room temperature in 5% (w/v) nonfat milk containing 0.05% Triton X-100. G₁₂ antibody was diluted 1:100 into ZO-1 hybridoma and incubated at 4 °C for 1.5 h and rinsed three times with 0.05% Triton X-100 followed by incubation with goat anti-rabbit Texas Red and goat anti-rat FITC-conjugated secondary antibodies (Pierce) at a 1:100 dilution. Slides were viewed on a Bio-Rad MRC-1024/2p confocal microscope, and images were processed using Photoshop software (Adobe, San Jose, CA).

Immunoprecipitation and Western Immunoblot Analysis-- wt₁₂ or QL₁₂ cells were cultured with or without doxycycline for 3 days. Whole cell lysates were obtained by scraping monolayers in buffer B (100 mM NaCl, 2 mM EDTA, 10 mM HEPES, pH 7.5, 1 mM NaVO₄, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) and brief sonication on ice. Antibodies against ZO-1 (rat) or G₁₂ (rabbit) were added to the lysate overnight at 4 °C. Beads were washed with buffer B, and proteins were eluted in SDS-PAGE sample buffer. Western blot analysis using chemiluminescence detection (Pierce) was performed as described previously (13).

Transepithelial Resistance (TER) and Paracellular Flux Measurements-- MDCK cells were plated on polycarbonate filters (Transwell, Costar) at confluent density (~2 × 10⁵ cells/cm²), and TER was measured at different time points using a Millipore ERS electrical resistance system. Measurements are expressed in ohm × cm² as a mean of the original readings after subtraction of background values. Paracellular flux was measured as described previously (14) using cells cultured on Transwell filters for 3 days with or without doxycycline. FITC-labeled 70-kDa dextran (concentration, 3.5 µM; Molecular Probes, Eugene, OR) was added into the apical chamber, and aliquots were taken from the lower chamber after 20, 40, and 60 min. FITC-dextran concentrations were determined using a fluorescent plate reader and standard curves (excitation, 485 nm; emission, 530 nm; Cytofluor 2300, Millipore Corp., Waters Chromatography, Bedford, MA) as described previously (14).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The function(s) for multiple ZO proteins within the TJ is unknown, but other PDZ family members have been shown to provide a scaffold for signaling complexes. In Drosophila, a G protein signaling complex has been identified in association with a related PDZ protein (15). We and others have demonstrated localization of signaling molecules within the TJ by confocal microscopy (7, 8, 16, 17), and other studies have shown immunoprecipitated ZO-1 and ZO-2 as a complex containing other proteins (9, 18, 19). To address whether heterotrimeric G subunits can directly interact with ZO-1 or ZO-2, we tested whether GST fusion proteins of G_s, G_i2, G_q, G₁₂, or G₁₃ could interact with in vitro translated ZO-1 and ZO-2. We found that only GST-G₁₂ consistently interacted with ZO-1 and ZO-2 in pull-down experiments (Fig. 1A). The failure to detect an interaction with the other G subunits could result from conformational constraints of fusion proteins or differences in protein stability. To confirm the G₁₂/ZO-1 interaction and identify the ZO-1 domain interacting with G₁₂, we tested in vitro translated G₁₂ and QL₁₂ for interaction with a series of GST fusion proteins containing various regions of ZO-1 (kindly provided by M. Balda, University of London). Fig. 1B shows ZO-1 domain structure and GST fusion proteins A-F. Fig. 1C compares the binding of in vitro translated G₁₂, QL₁₂, G_i2, and G₁₃ with GST alone, A fusion protein (amino acids 295-633, containing PDZ3 and SH3 domains), and B fusion protein (PDZ3 domain only). There was no detectable interaction of the in vitro translated G subunits with GST alone or the PDZ3 domain (Fig. 1C, B fusion protein). When normalized to the starting material, the A fusion protein interacted significantly better with G₁₂ and QL₁₂ (>10% bound, n = 3) than with G₁₃ or G_i2 (<1%). We next tested G₁₂ and QL₁₂ for interactions with C, D, E, and F fusion proteins and found that both G₁₂ subunits interacted with C and D GST fusion proteins, but there was no significant interaction with F fusion protein or with GST alone (Fig. 1D). In these experiments with in vitro translated proteins, we did not detect interactions with the E fusion protein (SH3). However, when these GST binding experiments were repeated with lysates from G₁₂ expressed in MDCK cells, there was a readily detectable interaction with the SH3 (E fusion protein) domain (see Fig. 2D). G₁₂ proteins expressed in vitro may have a lower affinity for ZO-1 than proteins expressed in cells due to the absence of lipid modifications. To see whether SH3 domains from other proteins could interact with G₁₂, we tested GST-SH3 domains from several other proteins (kindly provided by Dr. Bruce Mayer) and found no detectable interactions with in vitro translated G₁₂ (Fig. 1E).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Interaction of G subunits with ZO-1 and ZO-2. A, GST alone, Gi2-, and G12-GST fusion proteins (1 µg) were incubated overnight with in vitro translated [35S]methionine-labeled ZO-1 and ZO-2 as described under "Experimental Procedures." 1 µl of in vitro translated ZO-1 and ZO-2 are shown on the right; 10 µl of each were used in overnight incubations, and the autoradiogram was exposed for 72 h. B, structure of ZO-1 with PDZ domains, guanylate kinase (GUK), SH3, acidic, , and proline-rich domains. GST constructs A-F with specific amino acids and relationship to overall structure are shown underneath. C, GST, ZO-1-A-, and ZO-1-B-GST fusion proteins (1 µg) were incubated with 10 µl of [35S]methionine-labeled wt13, wti2, wt12, and QL12 as described under "Experimental Procedures." 1 µl of each in vitro translated protein is shown on the right. A discrete separation of these G subunits was consistently seen under these electrophoresis conditions. Exposure time was 72 h. D, GST and fusion proteins C, D, E, and F (1 µg) were incubated with [35S]methionine-labeled wt12 and QL12 (10 µl) as described above. 1 µl of wt12 is shown in the first lane. Exposure time was 72 h. E, [35S]methionine-labeled wt12 was incubated with equivalent amounts of GST-ZO-1-A and GST-SH3 from Abl, Crk, n-Src, c-Src, and Grb2 and analyzed as described above. Exposure time was 72 h. IVT, in vitro translated.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   wt12 and QL12 expression, localization, and interactions with ZO-1 in MDCK cells. A, Western blot for G12 expression in wt12 and QL12 MDCK cells. Cells were cultured in the presence of 40 ng/ml doxycycline (+dox) and then switched to doxycycline-free medium (dox). Cell lysates were analyzed by Western blot after 24, 48, and 72 h. B, confocal co-localization of G12 and ZO-1 in MDCK cells. wt12 and QL12 MDCK cells ± dox were double stained with antibodies to G12 and ZO-1 and analyzed by confocal microscopy. Bar = 20 µM. C, ZO-1 Western blot of wt12 and QL12 MDCK cell lysates and G12 immunoprecipitations. Cells were cultured +dox or dox for 72 h and analyzed in parallel as described under "Experimental Procedures." Antibody-absent control (beads only) is shown on the right. Similar results were obtained in three independent experiments. D, interactions of wt12 and QL12 expressed in MDCK cells with ZO-1-GST fusion proteins. Approximately 10 µg of cell lysate was incubated with 1 µg of GST fusion proteins as described in Fig. 1. Samples were washed, eluted, and analyzed by SDS-PAGE followed by Western blot with G12 antibody. dox + indicates parallel analysis of lysates from cells cultured in doxycycline; no symbol indicates cells were grown in dox medium.

G₁₂ is difficult to study in cells due to its low levels of expression. Therefore, we established a model system in MDCK cells. Similar to other reports (12, 20), we utilized Tet-Off inducible MDCK cell lines to express wt₁₂ and QL₁₂. wt₁₂ and QL₁₂ proteins were expressed within 24-48 h after the removal of doxycycline (Fig. 2A). Protein levels remained constant for up to 10 days in the absence of doxycycline, and expression of G₁₂ proteins could be resuppressed within 48 h with the readdition of doxycycline (not shown). G₁₂ had previously been demonstrated to co-localize with ZO-1 in MDCK cells by confocal microscopy (16). Endogenous G₁₂ was barely detectable by Western blot and confocal microscopy in the presence of doxycycline (Fig. 2, A and panels a and e in B), but after 48 h of G₁₂ expression (dox) there was co-localization with ZO-1 in the TJ (Fig. 2B, panels b and d). Expression of QL₁₂ in MDCK cells resulted in an altered cell phenotype and a complex staining pattern for QL₁₂ and ZO-1 (Fig. 2B, panels f and h). Some QL₁₂ was localized intracellularly, and this could occur from overexpression and/or reduced affinity for the plasma membrane. Although the staining patterns of QL₁₂ and ZO-1 were different, there appeared to be partial overlap in the lateral margins of the cell. Consistent with this observation, immunoprecipitation of G₁₂ from both wild-type and QL-expressing cells co-precipitated ZO-1 (Fig. 2C). In the absence of G₁₂ expression (+dox) or with no added antibody (Fig. 2C, last lane) there was no detectable ZO-1. The expression levels of ZO-1 were not affected by wt₁₂ or QL₁₂ expression (determined by Western blot, not shown), and the increased immunoreactivity in QL lysates was unique to this experiment. We next utilized the ZO-1-GST fusion proteins (Fig. 1B) and Western blots to determine whether wt₁₂ or QL₁₂ expressed in MDCK cells could bind to specific ZO-1 domains. The ZO-1 SH3 domain (E fusion protein) was capable of binding to both wt₁₂ and QL₁₂ from cell lysates prepared from dox cells (Fig. 2D). Identical to the in vitro results (Fig. 1), the B and F fusion proteins did not interact (not shown), and GST alone was negative (Fig. 2D). These results indicate that the SH3 domain is sufficient for interactions with G₁₂ but do not exclude contributions from other ZO-1 domains.

The disrupted appearance of ZO-1 in the lateral membrane of QL₁₂-expressing MDCK cells (Fig. 2B) suggested that barrier function might be altered in these cells. To address this question, we measured TER and paracellular flux. Base-line TER was comparable in MDCK Tet-Off cells, uninduced wt₁₂, and QL₁₂ MDCK cell lines (Fig. 3A, +dox at T = 0). There was some variability in base-line TER as seen with the two sets of QL₁₂ cells at T = 0, but this was comparable to other reports (20). Expression of wt₁₂ protein resulted in a small decrease in TER after 24 h but remained nearly identical to Tet-Off MDCK cells throughout the remainder of the experiment (Fig. 3A, open and closed squares). Although we cannot exclude a subtle role for overexpressing wt₁₂ on the junction, these effects did not correlate with wt₁₂ protein expression (±dox). However, inducing QL₁₂ expression consistently caused a significant fall in TER to ~25 ohm × cm² within 48 h (Fig. 3A, n = 7). This fall in TER was reversible as switching to +dox medium resulted in TER returning to normal over the next 24-48 h. There was a brief "overshoot" of base-line TER during TJ formation in QL₁₂ MDCK cells similar to what is typically observed during TJ assembly (21). The decrease in TER was sustained in QL₁₂ MDCK cells if doxycycline remained absent but could be reversed at any time by the readdition of doxycycline (not shown). We also measured paracellular flux of 70-kDa FITC-dextran in both G₁₂ MDCK cell lines cultured ± doxycycline for 3 days (Fig. 3B). Induction of QL₁₂ expression led to a greater than 3-fold increase in flux rate (12.8 ± 1.5-40.3 ± 10.4 pmol/min × cm², n = 7), while there was no significant change in wt₁₂ MDCK cells ± doxycycline (6.5 ± 1.4 versus 9.4 ± 2.5 pmol/min × cm², n = 7, Fig. 2B, left panel). As an additional control, we counted trypan blue-positive cells in QL₁₂ monolayers ± doxycycline and found no significant difference. Taken together, these results indicate that expression of QL₁₂, but not wt₁₂, reversibly increases paracellular permeability, and these observations are consistent with the changes in ZO-1 staining pattern seen in QL₁₂-expressing cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Paracellular properties of wt12 and QL12 MDCK cells. A, TER in MDCK cell lines. Transwell filters were cultured in +dox medium at T = 0. TER was measured every 24 h after switching to dox medium and again after switching back to +dox medium. Values are the mean ± S.E. for three determinations. Tet-Off MDCK cells are included for comparison (closed squares), and nearly identical results were obtained in five independent experiments. B, paracellular flux rate in wt12 and QL12 MDCK cells. Cells were cultured for 72 h ± dox, and the rate of movement for a 70-kDa FITC-dextran molecule was determined in triplicate. * indicates significant difference (p < 0.005).

In recent years, the identification of integral membrane proteins in the TJ has resulted in significant progress toward understanding the structural features of the barrier. Claudins are essential for barrier function (2) and for paracellular regulation of specific ions (22, 23). Claudin family members interact with the scaffolding proteins ZO-1, ZO-2, ZO-3, and MUPP-1 (4, 24), and there are multiple interactions of these scaffolding proteins with other tight junction proteins (occludin and junctional adhesion molecule, Ref. 25) and signaling molecules (26, 27). Several different signaling pathways regulate the barrier of intact epithelia. Activation and overexpression of protein kinase C isoforms or monomeric G proteins results in disruption of junctions and increased leakiness in kidney epithelial cells (12, 21, 28, 29). Furthermore, we have previously shown that activated G_i2 and G_s increase base-line TER in MDCK cells (7, 8). The finding that activation of G₁₂ lowers TER and increases paracellular permeability raises the possibility that multiple G proteins function in concert to regulate base-line TJ properties.

For the first time, we have demonstrated that ZO-1 directly interacts with a G protein  subunit and propose that ZO-1 functions to scaffold G₁₂. ZO proteins may organize signaling complexes within a discrete membrane microdomain, the TJ. Considering the complex signaling pathways that regulate the TJ, we would suggest that there are likely to be other direct interactions between signaling proteins and ZO proteins. We show that G₁₂ can also interact with ZO-2 (Fig. 1A), and other lower affinity interactions may not have been detected. The finding that wt₁₂ and QL₁₂ interact similarly with ZO-1 suggests that the interaction is independent of G₁₂ conformation and supports the notion that ZO-1 scaffolds G₁₂. Although we cannot exclude the possibility that ZO-1 directly regulates G₁₂, we found that the GST-ZO-1-A fusion protein (Fig. 1B) has no effect on GTPS binding to G₁₂.² The mechanism(s) for G₁₂ regulation of paracellular properties within the TJ and the role of the interaction with ZO-1 remain to be determined. One possibility is that through its interaction with ZO-1, G₁₂ is placed within close proximity to other regulatory proteins that also bind to ZO-1. Our experiments were not designed to distinguish between a role for the ZO-1/G₁₂ interaction and other possible G₁₂ downstream signaling pathways (such as through Rho). G₁₃ (67% amino acid identity to G₁₂), did not interact with ZO-1 or ZO-2. Since G₁₃ shares some downstream pathways with G₁₂, a comparison of G₁₃ and G₁₂ cell lines may permit us to distinguish the role of ZO-1 binding from other mechanisms regulating the junction.

Our results indicate that the SH3 domain of ZO-1 is sufficient for binding to G₁₂, and there may be a putative G₁₂ binding motif contained within the ZO-1 SH3 domain. The cytoplasmic domain of E-cadherin binds to G₁₂ and G₁₃, and although E-cadherin is expressed in the adherens junction, this interaction regulates association of the transcriptional activator -catenin (30). Recently a cluster of charged amino acids within the C-terminal domain of E-cadherin has been shown to be required for G₁₂ binding (amino acids 854-864, Ref. 31). Comparison of these amino acids with the SH3 domain of ZO-1 identifies a highly conserved group of charged residues at amino acids 514-518 (EX(D/E)K(D/E)). The SH3 domain of ZO-2 contains an identical sequence, and ZO-2 also binds to G₁₂ (Fig. 1A). However, the SH3 domain of ZO-3 is lacking the last two charged amino acids, and we were unable to detect an interaction of in vitro translated ZO-3 with G₁₂ (results not shown). Finally, a recent report identified an interaction between G₁₂ and Hsp90, and Hsp90 also contains this identical cluster of charged residues (32). Other motifs have also been identified for G₁₂ binding (33). Taken together, these findings support a scaffolding function for ZO-1 and suggest a similar role for ZO-2 and ZO-3 within the TJ. Unraveling the specific connections between ZO proteins and other signaling molecules will help provide the keys to understanding TJ regulation.

	ACKNOWLEDGEMENTS

We are indebted to Dr. Maria S. Balda (University of London) and Dr. Bruce Mayer (Children's Hospital) for sharing ZO-1 and SH3 constructs, respectively. We thank Michelle Lowe for excellent technical assistance with the confocal microscope and Sean Colgan for help with the flux measurements.

	FOOTNOTES

^* 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.

^§ Supported by Grant Me 1760/1-1 from the Deutsche-Forschungs-gemeinschaft, Bonn, Germany.

Supported by the Daimler-Benz Foundation, Ladenburg, Germany.

^** Supported by National Institutes of Health Grant GM55223 and a clinical scientist award from the National Kidney Foundation. To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker@rics.bwh.harvard.edu.

Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.C200240200

² B. Denker, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: TJ, tight junction; ZO, zonula occludens; SH3, Src homology 3; GST, glutathione S-transferase; QL, Q229L; wt, wild type; MDCK, Madin-Darby canine kidney; dox, doxycycline; FITC, fluorescein isothiocyanate; TER, transepithelial resistance; GTPS, guanosine 5'-3-O-(thio)triphosphate; Tet, tetracycline.

	REFERENCES

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				T. E. Meigs, J. Juneja, C. T. DeMarco, L. N. Stemmle, D. D. Kaplan, and P. J. Casey Selective Uncoupling of G{alpha}12 from Rho-mediated Signaling J. Biol. Chem., May 6, 2005; 280(18): 18049 - 18055. [Abstract] [Full Text] [PDF]

				D. Zhu, K. S. Kosik, T. E. Meigs, V. Yanamadala, and B. M. Denker G{alpha}12 Directly Interacts with PP2A: EVIDENCE FOR G{alpha}12-STIMULATED PP2A PHOSPHATASE ACTIVITY AND DEPHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN, Tau J. Biol. Chem., December 31, 2004; 279(53): 54983 - 54986. [Abstract] [Full Text] [PDF]

				K. T. Kahle, G. G. MacGregor, F. H. Wilson, A. N. Van Hoek, D. Brown, T. Ardito, M. Kashgarian, G. Giebisch, S. C. Hebert, E. L. Boulpaep, et al. Paracellular Cl- permeability is regulated by WNK4 kinase: Insight into normal physiology and hypertension PNAS, October 12, 2004; 101(41): 14877 - 14882. [Abstract] [Full Text] [PDF]

				D. D. Mruk and C. Y. Cheng Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocr. Rev., October 1, 2004; 25(5): 747 - 806. [Abstract] [Full Text] [PDF]

				E. E. Schneeberger and R. D. Lynch The tight junction: a multifunctional complex Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1213 - C1228. [Abstract] [Full Text] [PDF]

				T. N. Meyer, J. Hunt, C. Schwesinger, and B. M. Denker G{alpha}12 regulates epithelial cell junctions through Src tyrosine kinases Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1281 - C1293. [Abstract] [Full Text] [PDF]

				A. A. Waheed and T. L. Z. Jones Hsp90 Interactions and Acylation Target the G Protein Galpha 12 but Not Galpha 13 to Lipid Rafts J. Biol. Chem., August 30, 2002; 277(36): 32409 - 32412. [Abstract] [Full Text] [PDF]