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 adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

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-...

VE-cadherin was first identified in the early 1990s and quickly emerged as an important endothelial cell adhesion molecule. The past decade of research has revealed key roles for VE-cadherin in vascular permeability and in the morphogenic events associated with vascular remodeling. The details of how VE-cadherin functions in adhesion became apparent with structure-function analysis of the cadherin extracellular domain and with the identification of the catenins, a series of cytoplasmic proteins that bind to the cadherin tail and mediate interactions between cadherins and the cytoskeleton. Whereas early work focused on the armadillo family proteins beta-catenin and plakoglobin, more recent investigations have identified p120-catenin (p120(ctn)) and a related group of armadillo family members as key binding partners for the cadherin tail. Furthermore, a series of new studies indicate a key role for p120(ctn) in regulating cadherin membrane trafficking in mammalian cells. These recent studies place p120(ctn) at the hub of a cadherin-catenin regulatory mechanism that controls cadherin plasma membrane levels in cells of both epithelial and endothelial origin.

p120-catenin (p120) has emerged over the past several years as an important regulatory component of the cadherin adhesive complex. A core function of p120 in mammalian cells is to stabilize cadherins at the cell membrane by modulating cadherin membrane trafficking and degradation. In this way, p120 levels act as a set point mechanism that tunes cell-cell adhesive interactions. The primary control point for this regulatory activity appears to be at the level of cadherin internalization from the plasma membrane, although p120 may also impact other aspects of cadherin trafficking and turnover. In the following review, the general mechanisms of cadherin trafficking are discussed, and models for how p120 may influence cadherin membrane dynamics are presented. In one model, p120 may function as a "cap" to bind the cadherin cytoplasmic tail and prevent cadherin interactions with endocytic membrane trafficking machinery. Alternatively, p120 may stabilize cell junctions or regulate membrane trafficking machinery through interactions with small GTPases such as Rho A, Rac and Cdc42. Through these mechanisms p120 exerts influence over a wide range of biological processes that are dependent upon tight regulation of cell surface cadherin levels.