The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

Abstract

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins. INTRODUCTIONTight junctions (TJ) are a hallmark of polarized epithelial cells, providing the paracellular barrier required to separate tissue spaces (Van Itallie and Anderson, 2004), contributing to maintenance of apical-basolateral cell polarity and providing a site for cell-cell signaling (Schneeberger and Lynch, 2004;Matter et al., 2005;Shin et al., 2006). At the ultrastructural level, tight junctions appear as highly organized strands that encircle the apical-lateral boundary. These strands are composed of transmembrane proteins such as occludin, tricellulin, and one or more members of the claudin family, which create the paracellular barrier. These proteins are, in turn, associated with a cytosolic plaque of proteins that is closely associated with the cortical cytoskeleton. The sequence of events that bring about the proper continuous apical localization of TJ proteins remains unknown. One approach to this problem has been to define components in upstream pathways required to induce cell polarity (Shin et al., 2006). Another approach has been to elaborate the biochemical interactions among the more than 40 transmembrane and cytosolic components of the TJ and to define their functional role in somatic cells using gene ablation, transcriptional inhibition, or dominant negative strategies (Schneeberger et al., 2004). Despite considerable efforts by many groups, the precise mechanism of junction assembly is poorly understood.The cytoplasmic protein ZO-1 is proposed to be one of the key regulators of TJ assembly (reviewed in Fanning, 2006). ZO-1 is member of a large family of membrane-associated scaffolding and signaling molecules known as the membrane-associated guanylate kinase homologues (MAGUKs). These proteins are characterized by a core motif of conserved protein-binding domains including one or more PDZ domains, an SH3 domain and a GUK domain (Funke et al., 2005). By analogy to other MAGUKs,...

Effects of prednisolone on the dystrophin-associated proteins in the blood–brain barrier and skeletal muscle of dystrophic mdx mice

“…48 The main molecular components of the TJs are transmembrane proteins, as occludin and claudins link to adaptor cytoplasmic proteins such as ZO-1. [49][50][51] For both occludin and ZO-1, phosphorylation levels help regulate barrier permeability, [52][53][54] and increased paracellular permeability associated with the tyrosine phosphorylation, and dissociation of occludin and ZO-1 from the cytoskeleton can be caused by oxidative stress. 55 Furthermore, vascular endothelial growth factor (VEGF) can increase intercellular permeability by enhancing ZO-1 and occludin phosphorylation, causing displacement and the reduction of ZO-1 expression, 56,57 corroborating the role of a tyrosine kinasedependent mechanism for the association of the junctional complex to cytoskeleton.…”

ZO-1- and ZO-2-Dependent Integration of Myosin-2 to Epithelial Zonula Adherens

“…In epithelial cells, ZO-1/2 are exclusively located at the zonula occludens (ZO) composed of tight junctions (TJs); in the absence of the ZO, they are enriched at the cell-cell adherens junctions (AJs) (Stevenson et al, 1986;Itoh et al, 1993). In epithelial cells lacking ZO-1/2, evidence has accumulated suggesting that ZO-1/2 play critical roles in the formation and function of epithelial-typed fully linearized AJs and TJs, i.e., the zonula adherens (ZA) and ZO, respectively McNeil et al, 2006: Fanning et al, 2007Hernandez et al, 2007;Ikenouchi et al, 2007). In this respect, ZO-1/2 reportedly bind to AJ-and TJ-constitutive proteins (Itoh et al, 1997(Itoh et al, , 1999aYamamoto et al, 1997;Schmidt et al, 2004).…”

The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

“…The SH3 domain is required for localization of ZO proteins to the TJ, in a process that is not yet understood (20), whereas the GUK domain binds to the transmembrane protein occludin. Although it is not known how the activity of these different domains is coordinated during TJ assembly, it is known that the PSG core motif is a necessary component of assembly and that elements within the core motif regulate the positioning of TJ proteins within the lateral plasma membrane (17,20). Thus, the PSG core motif is a critical element of TJ assembly.…”