RAC1 Regulates Adherens Junctions through Endocytosis of E-Cadherin

Abstract

The establishment of cadherin-dependent cell-cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell-cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin-mediated cell-cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell-cell contacts. The ability of Rac1 to disrupt cell-cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell-cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin-catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell-cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrinbiotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.

A Ca2+-dependent signalling circuit regulates influenza A virus internalization and infection

“…3g,h), indicating that RhoA promotes both CME and CIE. On the other hand, Rac1 expression affected only the uptake of dextran (Fig. 3g,h), suggesting that Rac1 regulates CIE but not CME, consistent with previous observations 34,35 . We examined the molecular hierarchy among RhoA, Rac1 and Ras.…”

RhoA GTPase Regulates M-Cadherin Activity and Myoblast Fusion

“…M-cadherin ubiquitination and endocytosis in RhoAV14-expressing cells might result from its failure to associate with p120 catenin (Figure 10). Indeed, endocytosis has emerged as a regulatory mechanism that modulates cadherin cell surface levels in cells (Le et al, 1999;Akhtar and Hotchin, 2001;Palacios et al, 2002), and recent reports have shown that p120 catenin controls VE-cadherin internalization and degradation (Xiao et al, 2003). Thus, p120 is a regulator of cell-cell adhesion through the maintenance of cadherin levels in cells (Ireton et al, 2002;Davis et al, 2003;Peifer and Yap, 2003).…”

EMT, the cytoskeleton, and cancer cell invasion

“…Moreover, integrinactivated focal adhesion kinase (FAK) can phosphorylate β-catenin and thus induce its ubiquitylation and degradation and the disassembly of the E-cadherin cell adhesion complex [59]. Endocytosis of E-cadherin can occur via clathrin or caveolin-dependent mechanisms [60][61][62]. A key player in clathrin-mediated E-cadherin endocytosis is Arf6, a Ras-related small GTPase.…”