The E-cadherin/catenin complex regulates Ca++-dependent cell–cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with β-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks β-catenin binding is also localized intracellularly. Thus, β-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that β-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that 13-catenin is the linker protein between E-cadherin and c-catenin and that E-cadherin does not bind directly to a-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of a-catenin are independent binding sites for 8-catenin. In addition to 13-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike 13-catenin, p120does not bind a-catenin in vitro, although a complex of p120 and endogenous oi-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and x-catenin revealed two catenin pools in cell lysates: an -1000-to '2000-kDa complex bound to E-cadherin and an -220-kDa pool that did not contain E-cadherin. Only 18-catenin in the -220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.The cadherin superfamily comprises glycoproteins responsible for calcium-dependent, homotypic cell interactions (1). Ecadherin is generally expressed in epithelial tissues and has been shown to regulate cell-cell adhesion (2), cell migration (3), morphogenesis (4), and the establishment of membrane polarity (5).Homotypic interactions between extracellular domains of cadherins are necessary but not sufficient for cell-cell adhesion (2). Linkage of the cadherin cytoplasmic domain to three cytosolic proteins, named a-catenin, ,B-catenin, and plakoglobin (y-catenin), is required (6-8). Although a-catenin, 3-catenin, and plakoglobin can be coimmunoprecipitated in a complex with E-cadherin (1, 7), the binding order of proteinprotein interactions has not been resolved. Insight into this problem is important for understanding functions of the cadherin-catenin complex and the regulation of cadherincatenin complex assembly. Here, we define the binding order of protein-protein interactions in the cadherin-catenin complex using genetic and biochemical approaches. MATERIALS AND METHODSStrain and Microbiological Techniques. All cloning procedures and bacterial transformation were performed by standard procedures outlined by Sambrook et at (9). Yeast strain Y190 (MATa gal4 gal80 his3-200 trpl-901 ade2-101 ura3-52

Background: MGP inhibits tissue calcification, but underlying mechanisms are understudied. Results: In MGP null mice, TG2 ablation prevents calcifying cartilaginous vascular lesions but does not affect elastocalcinosis and elastin fragmentation associated with increased elastase adipsin. Conclusion: MGP acts via two distinct mechanisms. Significance: Our study identifies TG2 and adipsin as potential therapeutic targets in vascular disease linked to MGP deficiency.

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N-terminal mutations in ␤-catenin that inhibit ␤-catenin degradation are found in primary tumors and cancer cell lines, and increased ␤-catenin͞T cell factor (TCF)-activated transcription in these cells has been correlated with cancer formation. However, the role of mutant ␤-catenin in cell transformation is poorly understood. Here, we compare the ability of different N-terminal mutations of ␤-catenin (⌬N131, ⌬N90, ⌬GSK) to induce TCF-activated transcription and anchorage-independent growth in Madin-Darby canine kidney epithelial cells. Expression of ⌬N90 or ⌬GSK ␤-catenin increased TCF-activated transcription but did not induce significant anchorage-independent cell growth. In contrast, deletion of the ␣-catenin-binding site in ⌬N131 ␤-catenin reduced TCF-activated transcription, compared with that induced by ⌬N90 or ⌬GSK ␤-catenin, but significantly enhanced anchorage-independent cell growth.