Dimerization of the Scaffolding Protein ZO-1 through the Second PDZ Domain

Abstract

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH2-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the β-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3. The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH2-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the β-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3. The tight junction (TJ) 2The abbreviations used are: TJ, tight junction; MDCK, Madin-Darby canine kidney cells; SH, Src homology; PBS, phosphate-buffered saline; ZO, zonula occludens; SEC-MALS, size exclusion chromatography with multiangle light scattering; GFP, green fluorescent protein; LS, light scattering; RI, refractive index. is the apical-most element of the junctional complex in epithelial and endothelial cells. This complex includes the TJ, adherens junctions, desmosomes, and gap junctions. The TJ forms a major barrier to paracellular movement of substances preserving unique chemical composition of apical and basolateral fluid compartments on opposite sides of the epithelial cell layer and coordinates transmembrane signaling (1Matter K. Balda M.S. Nat. Rev. Mol. Cell Biol. 2003; 4: 225-236Crossref PubMed Scopus (731) Google Scholar, 2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 2004; 286: C1213-C1228Crossref PubMed Scopus (1155) Google Scholar). The TJ is known to contain at least four classes of transmembrane proteins and >30 cytoplasmic signaling, scaffolding, and cytoskeletal proteins (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar). To understand how the barrier and signals are coordinated, more information is required on the specific molecular interactions of TJ proteins and specifically the proteins that coordinate interaction among transmembrane and cytosolic proteins. Peripheral membrane proteins of the tight junction are believed to be highly organized within a complicated network interconnecting cytoskeleton with transmembrane proteins. The three homologous zonula occludens (ZO) MAGUK proteins (ZO-1, ZO-2, and ZO-3) are among the best characterized peripheral membrane proteins of the tight junction. They are composed of three PDZ domains, an SH3-GUK unit, and an actin-binding region; these proteins are believed to cluster different components of tight junctions together through the direct binding to specific domains and/or regions (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar). Each has multiple known binding partners; some are shared among all three, such as binding to actin and claudins, whereas others are unique, such as binding of E4-ORF1 protein from type 9 adenovirus to ZO-2 (4Glaunsinger B.A. Weiss R.S. Lee S.S. Javier R. EMBO J. 2001; 20: 5578-5586Crossref PubMed Scopus (76) Google Scholar). In tissues lacking tight junctions, ZO proteins are localized to adherence junction where they are presumed to interact with cadherin-catenin complexes. Furthermore, ZO-1 was shown to bind many different connexins, structural components of gap junction, in a number of reports (5Toyofuku T. Yabuki M. Otsu K. Kuzuya T. Hori M. Tada M. J. Biol. Chem. 1998; 273: 12725-12731Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 6Kausalya P.J. Reichert M. Hunziker W. FEBS Lett. 2001; 505: 92-96Crossref PubMed Scopus (133) Google Scholar, 7Nielsen P.A. Baruch A. Shestopalov V.I. Giepmans B.N.G. Dunia I. Benedetti E.L. Kumar N.M. Mol. Biol. Cell. 2003; 14: 2470-2481Crossref PubMed Scopus (103) Google Scholar). Much efforts has been made to define the function of ZO proteins, for example through exogenous expression of truncated ZO-1 (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 8Ryeom S.W. Paul D. Goodenough D.A. Mol. Biol. Cell. 2000; 11: 1687-1696Crossref PubMed Scopus (74) Google Scholar, 9Reichert M. Muller T. Hunziker W. J. Biol. Chem. 2000; 275: 9492-9500Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and ZO-3 (10Wittchen E.S. Haskins J. Stevenson B.R. J. Cell Biol. 2000; 151: 825-836Crossref PubMed Scopus (53) Google Scholar). A high degree of sequence conservation between ZO proteins suggests that these proteins may have a significant functional redundancy, making it difficult to elucidate their function. Strong evidence that ZO proteins are indeed functionally redundant came from a recent report demonstrating that in ZO-1-deficient cells, cytoplasmic ZO-2 was recruited into tight junctions to compensate for the loss of ZO-1 (11Umeda K. Matsui T. Nakayama M. Furuse K. Sasaki H. Furuse M. Tsukita S. J. Biol. Chem. 2004; 279: 44785-44794Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). ZO proteins are known to hetero-oligomerize via their second PDZ domains. ZO-1 binds to both ZO-2 and ZO-3, whereas ZO-2 and ZO-3 do not bind each other (12Wittchen E.S. Haskins J. Stevenson B.R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Because no endogenous ZO-1 from MDCK cells was detected in immunoprecipitates of transiently transfected human ZO-1, it was concluded that ZO-1 does not form homo-oligomers (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar). Hence, ZO proteins are thought to exist exclusively as ZO-1/ZO-2 and ZO-1/ZO-3 heterodimers (12Wittchen E.S. Haskins J. Stevenson B.R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Determining whether homodimers exist is critical for understanding the complexity of protein networks that ZO proteins could create. In this work we demonstrate that, contrary to the previous beliefs, ZO-1 forms a stable dimer via its second PDZ domain and partially characterizes biophysical properties of the dimer. We also show that significant amounts of ZO-1 in MDCK cells exist as a homodimer. Antibodies and Affinity Media—Mouse monoclonal antibodies were purchased from Molecular Probes Inc. (Eugene, OR). R40.76 antibody was obtained from Bruce Stevenson (Salk Institute, La Jolla, CA). All other antibodies and goat anti-rat IgG immobilized on Sepharose 4B were purchased from Zymed Laboratories Inc. Protein G-Sepharose was purchased from GE Healthcare. Molecular Cloning—A NotI-XhoI fragment of plasmid pSKZO-1 encoding about 1000 amino acid residues of ZO-1 was cloned into the pFastBacHTa vector. The resulting plasmid was digested with NheI and HindIII and ligated by PCR with a fragment of ZO-1 (∼720 bp) amplified with primers AAT CAT AAG GAG GTA GAA CGA GGC and TTT AA GCT TCT AAT CCG CCT TTC CCT CGG AAA CCC to introduce a stop codon after amino acid 806. To modify the NH2 terminus, the construct was digested with NotI and KasI and ligated with annealed oligonucleotides GCG CCA TGT CCG CCA GAG CTG C and GGC CGC AGC TCT GGC GGA CAT G. The resulting construct encodes amino acids 1-806 of human ZO-1 with an NH2-terminal His6 tag. Other constructs were obtained by similar procedures (details are available upon request). These constructs and their amino acid sequence ranges are presented in Fig. 4. GFP-ZO-1 was obtained by subcloning of ZO-1 into pEGFP-C1 vector, followed by subcloning of GFP-ZO-1 into pIRES vector. Expression and Purification of Recombinant ZO-1 Fragments— Recombinant bacmids and baculoviruses were obtained using Bac-to-Bac kit (Invitrogen). High Five cells were infected at a multiplicity of infection of 3-5, harvested 48-60 h after infection, and kept frozen at -80 °C. Cell pellets were thawed in 6 volumes of lysis buffer (20 mm Tris-Cl, pH 8, 150-500 mm NaCl, 10% glycerol, 0.8% Igepal CA-630, 12.5 mm imidazole, 5 mm 2-mercaptoethanol, and Complete™ protease inhibitor mixture (Roche Applied Science)), homogenized in a Dounce homogenizer, and centrifuged for 10 min at 20,000 × g. Protein was purified from the supernatant using EZview™ Red His-Select HC nickel affinity gel from Sigma and eluted into PBS containing 5 mm β-mercaptoethanol and 125 mm of imidazole. MDCK Cell Culture, Transfection, and Stable Clone Selection—MDCK cells were cultivated in DMEM containing 10% fetal bovine serum using 25-cm2 tissue culture flasks or 6-well plates. Cells in 6-well plates were transfected with 3 μg of GFP-ZO-1 plasmid and 5 μg of Cellfectin (Invitrogen) according to the manufacturer's instructions. Transfected cells were cultivated in media containing 0.8 mg/ml G-418, and cell clones were isolated with cloning rings. Immunoprecipitation and Immunoblotting—MDCK cells grown on 50-cm2 Petri dishes were washed twice with PBS and lysed in 0.6 ml of RIPA buffer (20 mm Tris-Cl, 150 mm NaCl, 5 mm 2-mercaptoethanol, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS). The lysate was clarified by centrifugation at 15,000 × g for 10 min and added to 30 μl of protein G beads preloaded with 5 μg of antibodies. Beads were incubated with the lysates for 2 h, washed five times with PBS containing 0.2% of Triton X-100, and boiled for 5 min in 100 μl of electrophoresis sample buffer to elute bound proteins. Protein samples were resolved on 4-12% gradient polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose membranes. Membranes were blocked in PBS containing 5% nonfat dry milk, incubated with primary and fluorophore-labeled secondary antibodies, and scanned with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Analytical Centrifugation—Sedimentation equilibrium experiments were performed with a Beckman Optima XL-A analytical centrifuge. Partial specific volume of NZO-1 was estimated to be 0.727 using SEDNTERP software. NZO-1 at 2-6 μm was analyzed in PBS containing 1 mm of 2-mercaptoethanol at 20 °C at speeds ranging between 6,000 and 20,000 rpm. Samples were spun in a 6-channel 12-mm external fill equilibrium center-piece in an An-60 Ti rotor. Scans (280 nm) were collected at equilibrium in radial step mode with 0.001-cm steps and 5-point averaging. Data fitting was performed with one-component or monomer-dimer equilibrium models using XL-A/XL-I data analysis software (Beckman Instruments, Rockville, MD). Size Exclusion Chromatography with Multiangle Light Scattering (SEC-MALS)—SEC-MALS experiments were performed with an Akta FPLC (GE Healthcare) coupled to a Wyatt DAWN EOS light scattering instrument, Wyatt Optilab refractometer, and Wyatt dynamic light scattering module (Wyatt Technology Corp., Santa Barbara, CA). For chromatographic separations, a Superose 6 HR 10/30 column (GE Healthcare) equilibrated with phosphate-buffered containing 5 mm 2-mercaptoethanol was used at an of Molecular weight were made by software using a of The NH2-terminal of ZO-1 a in protein corresponding to the NH2-terminal half of ZO-1 three PDZ domains, an a and an domain was purified to from cells. NZO-1 as a on polyacrylamide gel in the of molecular of NZO-1 is no significant Size exclusion chromatography revealed that NZO-1 as a with an molecular of that NZO-1 may form a stable or be highly Because size exclusion chromatography data are on the of the the of the molecular weight of the protein is To the of NZO-1 in we performed size exclusion chromatography with of light scattering and refractive this an of the molecular weight of the protein of its and/or amino acid composition J. T. PubMed Scopus Google Scholar). chromatographic of NZO-1 obtained from and are shown in Fig. The molecular of the NZO-1 from and data was to be that NZO-1 is a dimer. The molecular is of the volume the of significant amounts of and these is a dimer. refractive and light scattering of the of molecular weight on The the are in in the In an to determine the of the the NZO-1 was characterized by analytical In the of equilibrium data to the of a dimer in The analysis of equilibrium data that can be obtained using a monomer-dimer were obtained with a using the molecular weight of the dimer we concluded that are that they be This the of of monomer-dimer a analysis of binding which that the be of the define which of NZO-1 is for we purified truncated forms of NZO-1 using the expression and we their by We a truncated form lacking the NH2-terminal amino acid residues of ZO-1 was to via their NH2 G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Other constructs lacking different domains are shown on Fig. All constructs could be and purified to Molecular of all proteins from the of on gels are to the corresponding for acids which has an molecular 10 All purified forms of ZO-1 were to in to determine their molecular to all proteins eluted as with or no of molecular weight on volume not The of experiments are in molecular of all truncated forms of ZO-1 construct was to be a are to the of corresponding Because all constructs the second PDZ these indicate that is necessary and sufficient for of dimerization motif of ZO-1 by light of of by light in a of ZO-1 in ZO-1 was not previously thought to we to determine whether exist in vivo. We stable of MDCK cells or As previously H. Cell Biol. PubMed Scopus Google the of GFP-ZO-1 was similar to endogenous ZO-1, both exclusively in tight junctions The of ZO-1 in cells ZO-1 was from and GFP-ZO-1 cell using the R40.76 monoclonal antibody antibody canine not exogenous human A significant of GFP-ZO-1 was to with endogenous ZO-1, the of ZO-1 or in In GFP-ZO-1 was with antibodies endogenous ZO-1 was also to with binding of to ZO-1 was in experiments To understand how ZO-1 is among and with ZO-2 and ZO-3, we performed a of to ZO-1, ZO-2, or ZO-3 from MDCK cell lysates with of the proteins in bound and The NH2-terminal of ZO-1 acids and the full-length ZO-2 in cells were used as to ZO-3 from cells could not be obtained in sufficient ZO-2 and ZO-3 were from the MDCK cell lysate to of ZO-1 is not bound to ZO-2 and ZO-3. A and as shown in Fig. show that of ZO-2 and of ZO-3 could be with antibodies analysis of ZO-1 revealed that about of ZO-1 is not bound to ZO-2 or ZO-3 and in the we that about of ZO-1 is bound to ZO-2 and ZO-3. we used MDCK cells stably GFP-ZO-1 to the of ZO-2 and GFP-ZO-1 that with endogenous Fig. that endogenous ZO-1 could be from MDCK cells stably with antibodies revealed that about of GFP-ZO-1 with endogenous with antibodies both GFP-ZO-1 and endogenous ZO-1 the amounts of these proteins. ZO-1 and GFP-ZO-1 were to be at Analysis of the protein sample obtained from R40.76 immunoprecipitates revealed that it of endogenous ZO-1 and about of Analysis with antibodies revealed that of ZO-2 with endogenous ZO-1 in MDCK cells stably GFP-ZO-1 ZO-1 was the tight junction protein to be and is thought to be of the major at tight junctions. The and biophysical properties of ZO-1 were characterized of the with the expression of constructs in Using expression we were to amounts of the NH2-terminal of ZO-1 ZO-1 with other MAGUKs like and which the of its biophysical Because of gel of we used multiangle light scattering to its NZO-1 was to exist exclusively as a stable dimer. Analytical centrifugation that NZO-1 forms a dimer with a submicromolar equilibrium dissociation constant. a that NZO-1 form homodimers in vivo. the of various of ZO-1, we could as a dimerization The dimerization does not to via the of by as in the of B.J. 1999; PubMed Scopus Google the of the regions not dimer is to a specific dimerization as for PDZ domains of Lee J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and Lee M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of ZO-1 does not to to by domain as previously Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google a construct containing and domain was to exist exclusively as a Because ZO-1/ZO-2 interaction was shown to the second PDZ domains of ZO-1 and ZO-2 (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, M. K. Tsukita S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google it that second PDZ domains of all ZO proteins an to This is by the that the second PDZ domains of ZO proteins are and between of human ZO-1 and previous reports that various of ZO-2 bound to ZO-1 in no binding was detected using purified proteins for in binding M. K. Tsukita S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). on our this is to the of ZO-2 to of ZO-1 and/or In this in binding with purified ZO proteins to be with experiments of proteins in the The previous report of (3Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar) suggests that ZO-1 does not form homo-oligomers in MDCK cells transiently transfected human ZO-1 and its not endogenous canine This from that of the A for the previous to the interaction is that of and/or the expression of transfected ZO-1 the of the To the of and expression we cloned GFP-ZO-1 into the pIRES that is to cells with high of GFP-ZO-1 to the tight junction. Using R40.76 antibody to endogenous ZO-1 and antibody to exogenous we could show that homodimers of ZO-1 exist in vivo. In we could about of ZO-2 and of ZO-3 from MDCK cell that the of ZO-2 is bound to ZO-1, about of the ZO-1 to be of ZO-2 or ZO-3. In in to ZO-2 and ZO-3, a substantial of ZO-1 is not in RIPA buffer and is not to be in a complex with ZO-2 and ZO-3. We do not have that ZO-1 is from and in a cell as a or experiments with cell GFP-ZO-1 indicate that are at least as as ZO-1/ZO-2 that purified NZO-1 exclusively as a dimer with submicromolar it is that ZO-1 of ZO-2 and ZO-3 in the cell via its second PDZ The of or between all ZO proteins have to be it is that the of all of homodimers and heterodimers be on the of expression of all ZO proteins. In this it is that the expression of ZO proteins from each other in various tissues that various of and heterodimers of ZO proteins may form in different For ZO-3 is in endothelial tight junctions A. M. A. M. Furuse M. Tsukita S. 2003; PubMed Scopus Google and the expression of ZO-1 and ZO-2 show different the D. J. S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, a significant of ZO-3 and of ZO-2 is localized to the of highly polarized cells, whereas ZO-1 is localized exclusively to the tight junction This that and of ZO proteins may also have ZO-1 and other MAGUKs are in of different proteins at through the direct binding to and domains. To MAGUKs via different For MAGUK was to through of NH2-terminal J. Cell 2003; PubMed Scopus Google Scholar). The of was to be to as a J. Cell 2003; PubMed Scopus Google Scholar). ZO proteins, which are with tight junction in epithelial cells, are unique among MAGUKs in their to form highly stable via the second PDZ is that the dimerization is highly for the function of ZO proteins as molecular and our be at understanding the of dimerization for the function of ZO proteins. We of for the with the analytical centrifugation and light scattering