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 NH 2 -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 ␤-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)2 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 (1, 2). The TJ is known to contain at least four classes of transmembrane proteins and Ͼ30 cytoplasmic signaling, scaffolding, and cytoskeletal proteins (3). 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 ...

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…Confocal immunofluorescence of HeLa cells stably expressing full-length untagged Cx43 (HeLa Cx43) revealed a typical arrangement of Cx43 plaques with peripherally associated endogenous ZO-1 (Figure 2A). When tagged with DsRed and transiently expressed in HeLa Cx43 cells, ZO-1 localization ( Figure 2B) remained qualitatively indistinguishable from the localization pattern of endogenous ZO-1 (Figure 2A), consistent with other studies using tagged ZO-1 constructs (McNeil et al 2006;Riesen et al 2002;Utepbergenov et al 2006). Both endogenous ZO-1 and transiently expressed DsR-ZO-1 exhibited edge localization that included regions of partial overlap with gap junctional Cx43 immunofluorescence (Figure 2A and B), indicating colocalization of the two molecules and the possibility of direct physical interaction through ZO-1 PDZ2 binding to the Cterminus of Cx43 (Giepmans and Moolenaar 1998).…”

“…This difference in patterning indicates that C-terminal tagging of Cx43 and removal of the PDZ2 domain from ZO-1 may elicit distinct effects, suggesting that ZO-1 distribution at plaque edges involves elements of the Cx43 C-terminus and ZO-1 PDZ2 domain that act independently of one another. Regardless of the mechanistic details, these results confirm that ZO-1 targets to Cx43 GJs independently of PDZ2-mediated interactions involving either homodimerization of ZO-1 (Fanning et al 2007;Utepbergenov et al 2006) or physical interaction between ZO-1 and Cx43, and hint at the existence of discrete ZO-1 complexes associated with GJ edges. The molecular composition of ZO-1 complexes juxtaposed with GJ edges is unclear, but preliminary colocalization data (not shown) suggest a prominent role for N-cadherin, which may participate in the delivery of ZO-1 to GJs.…”

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

“…In contrast, the recovery of tagged ZO-2 is higher in samples from the N-terminal BL-ZO-1 fusion protein compared with C-terminal fused ZO-1-BL protein (Fig. 4, top row, middle), consistent with its known interaction with PDZ2, which is closer to the N terminus (17). This same pattern is observed for another tight junction scaffolding protein, MAGI3 (Fig.…”

“…Its N-terminal half contains three PDZ domains, an SH3 2 domain, and a region with homology to guanylate kinase (15). The first PDZ domain is the binding site for the strand-forming claudin family of proteins (16); PDZ2 is the site for heterodimerization with the ZO-1 homolog, ZO-2 (17); and PDZ3 is the binding site for the adhesive Ig superfamily tight junction protein, JAM (junctional adhesion molecule) (18). The guanylate kinase domain is the binding site for occludin (19,20).…”

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

“…Parental MDCK cells and μ1B knockdown MDCK cells were obtained from Enrique Rodriguez-Boulan, Weill Cornell Medical College, New York (25,38). MDCK cells stably expressing ZO-1-GFP were a kind gift from Alan S. Fanning, University of North Carolina, Chapel Hill, NC (40). All cell lines were maintained in DMEM containing 10% (vol/vol) bovine growth serum (BGS), nonessential amino acids, L-glutamine, and penicillin/streptomycin.…”