The mechanisms by which catenins regulate cadherin function are not fully understood, and the precise function of p120 catenin (p120ctn) has remained particularly elusive. In microvascular endothelial cells, p120ctn colocalized extensively with cell surface VE-cadherin, but failed to colocalize with VE-cadherin that had entered intracellular degradative compartments. To test the possibility that p120ctn binding to VE-cadherin regulates VE-cadherin internalization, a series of approaches were undertaken to manipulate p120ctn availability to endogenous VE-cadherin. Expression of VE-cadherin mutants that competed for p120ctn binding triggered the degradation of endogenous VE-cadherin. Similarly, reducing levels of p120ctn using siRNA caused a dramatic and dose-related reduction in cellular levels of VE-cadherin. In contrast, overexpression of p120ctn increased VE-cadherin cell surface levels and inhibited entry of cell surface VE-cadherin into degradative compartments. These results demonstrate that cellular levels of p120ctn function as a set point mechanism that regulates cadherin expression levels, and that a major function of p120ctn is to control cadherin internalization and degradation.

VE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cad cyto ) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cad cyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cad cyto mutant. Chloroquine treatment also resulted in the accumulation of a VEcadherin fragment lacking the ␤-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2R-VE-cad cyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells.Endothelial adherens junctions are adhesive intercellular contacts that are crucial for the maintenance and regulation of normal microvascular function (1-3). Alterations in adherens junction assembly influence endothelial cell motility, vascular morphogenesis, and permeability. Moreover, recent studies indicate that components of adherens junctions also function in intracellular signaling, leading to the current view that these complexes are plasma membrane domains that integrate chemical and mechanical signaling information (4). The major cellcell adhesion molecule at endothelial adherens junctions is VE-cadherin, a cadherin family member that is specifically expressed in endothelial cells (5). The cytoplasmic tail of the classic cadherins, including VE-cadherin, comprises two wellcharacterized domains. The juxtamembrane domain (JMD) 1 binds to the catenin p120, an armadillo family protein that is thought to regulate cadherin adhesive interactions by modulating the activity of Rho family GTPases (6 -8). At the carboxyl-terminal region of the cadherin cytoplasmic tail, a domain termed the catenin binding domain (CBD) interacts with ␤-catenin or plakoglobin (9). ␤-Catenin and plakoglobin both interact with ␣-catenin, which links cadherins to the actin cytoskeleton and to other actin-binding proteins such as ␣-actinin (10 -13). VE-cadherin also associates with the vimentin cytoskeletal network in endothelial cells through interactions with plakoglobin and the intermediate filament-binding protein desmoplakin (14). These unique intercellular junctions, containing both actin and vimentin-binding proteins, have been referred to as complexus adhaerentes (15-...

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG⅐Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin-and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin-and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.Desmosomes are adhesive junctions that provide robust adhesion between epithelial cells (1, 2). These organelles are prominent in tissues that experience substantial mechanical stress such as the heart, bladder, gastrointestinal mucosa, and skin. Desmosomes are comprised primarily of proteins from three major families, the desmosomal cadherins desmogleins and desmocollins, armadillo proteins such as plakoglobin and the plakophilins, and members of the plakin family of cytolinkers such as desmoplakin (1-3). Together, these proteins contribute to tissue integrity by coupling adhesive interactions mediated by the desmosomal cadherins to the keratin intermediate filament cytoskeleton, thereby integrating adhesive and cytoskeletal networks throughout the cells in a tissue. Although critical for tissue integrity, desmosomes are often remodeled and contribute to dynamic processes during development and wound healing. Furthermore, desmosomal components may also play pivotal roles in keratinocyte differentiation, morphogenesis, and tissue patterning as well as epithelial-mesenchymal transitions (4, 5).Pemphigus vulgaris (PV) 2 is a potentially fatal autoimmune skin disease in which autoantibodies are generated against the desmosomal cadherin, desmoglein-3 (Dsg3) (6 -8). Dsg3, a 130-kDa glycoprotein, is found primarily in the spinous and basal layers of the epidermis and throughout the oral mucosa (9). As a result, PV is characterized histologically by suprabasal loss of cell-cell adhesion (acantholysis) and clinically by blistering of the skin and erosion of mucous membranes (7,8). A wide range of approaches have demonstrated that Dsg3 is the key target of PV IgG (10, 11). In addition, experimentally generated mice in which the Dsg3 ge...

Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupt adhesion is not fully understood. To address this issue, primary human keratinocytes and patient IgG were utilized to define the morphological, biochemical and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living keratinocytes demonstrated that PV IgG cause rapid Dsg3 internalization which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde fashion along these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.

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