title: 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 beta-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.
authors.affiliation: Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599-7545, USA.
authors.forename: Darkhan I
authors.initials: DI
authors.lastname: Utepbergenov
authors.forename: Alan S
authors.initials: AS
authors.lastname: Fanning
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
mesh_terms: D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004285:Dogs; D008565:Membrane Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019108:Tight Junctions / Q000737:chemistry / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein