Cadherin cell-cell adhesion proteins play central roles in both the establishment (morphogenesis), and maintenance (tumor suppression), of a cell's differentiated state. Cadherins are present in all vertebrate solid tissues, and may be common to invertebrate tissues as well. The highly conserved cytoplasmic domains of cadherins associate with three (thus-far) identified polypeptides named alpha-catenin, beta-catenin, and plakoglobin (gamma-catenin), which are essential to cadherin-mediated cell-cell adhesion. alpha-Catenin and beta-catenin have also recently been found associated with the intracellular tumor-suppresser gene product APC, thus increasing the complexity of the catenins' known interactions and potential functions. beta-Catenin and plakoglobin are distinct proteins found within the same cells, but are highly homologous to each other in structure and amino acid sequence. Their specific versus shared structure-functions have not yet been determined, but the catenins collectively are thought to mediate the cadherins' (and APC's? interaction with the actin cytoskeleton - and likely with other intracellular proteins that we wish to identify. We have also recently obtained evidence suggesting that beta-catenin participates in the Wnt developmental signaling pathway. Our research is guided by three Specific Aims: A) Define the structure-function of beta-catenin. B) Define the structure-function of beta-catenin. C) Discover and characterize additional cytoplasmic proteins that interact with cadherins or catenins. In meeting these objectives, the experimental systems employed will include Xenopus eggs and embryos, cultures of canine MDCK and Xenopus A6 cell lines, and the yeast """"""""two-hybrid"""""""" system. Epitope-tagged mutant constructs of beta-catenin and plakoglobin, and beta-catenin-plakoglobin chimeric constructs, will be transfected into MDCK and A6 tissue culture cells, and the resulting phenotypes evaluated using phase-contrast and immunofluorescence microscopy. Mutant plakoglobin constructs will also be tested in Xenopus embryos, to examine its structure-function (relative to that of beta-catenin) in a developmental context. Immunoprecipitation of the epitope-tagged beta-catenin and plakoglobin constructs will permit identification of intracellular interactions leading to the observed phenotypes. Novel proteins interacting with the cadherin-catenin complex will be identified by using chemical cross-linker and yeast two-hybrid approaches.
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