Cadherins are a family of cell surface glycoproteins that mediate Ca2+- dependent, homophilic cell adhesion and thereby control tissue morphogenesis. All cadherins, reported thus far, contain a highly conserved cytoplasmic region that is crucially important for their function in cell adhesion by providing a linkage with the actin-based cytoskeleton. A member of the cadherin family, T-cadherin (T=truncated) was discovered in the developing chicken nervous system in this laboratory. T-cadherin lacks the cytoplasmic region and is anchored to the cellular plasma membrane through a glycosyl phosphatidyl inositol (GPI). Despite its different membrane anchor, T-cadherin is able to mediate Ca2+-dependent, homophilic cell adhesion in cellular aggregation assays. Thus, the function of T- cadherin must depend on a mechanism notably different from that of other cadherins.
The aim of this proposal is to dissect the mechanism of T- cadherin mediated cell adhesion. The first step is to determine whether T- cadherin is localized to adherens type junctions like other cadherins or to different membrane subdomains. To do this, the localization of T-cadherin expressed in heterologous fibroblasts by DNA transfection will be determined. T-cadherin will also be localized in two cell populations of the chicken eye that express endogenous T-cadherin: Lens cells that form specialized epithelial type junctions and neural retina cells which form synaptic specializations. Second, to determine which domains of T-cadherin are necessary for its function in cell adhesion and subcellular distribution, chimeric molecules will be generated in which selected regions of T-cadherin are interchanged with the corresponding domains of N- cadherin. A). The domain for homophilic T-cadherin binding will be mapped by generating chimeric molecules that have a T-cadherin binding specificity and the membrane attachment of N-cadherin. This analysis is important since T-cadherin lacks a peptide, HisAlaVal, that contributes to the homophilic binding function of other cadherins. B). The region in T- cadherin that functionally substitutes for the cytoplasmic domain of other cadherins will be defined by generating chimeric molecules with a N- cadherin binding specificity and the GPI-membrane anchor of T-cadherin. This region may be as short as the sequences required for the attachment of the GPI-anchor or extend to sequences anywhere between EC2 and the carboxy terminus of T-cadherin. Lastly, membrane molecules putatively associated with and required for the function of T-cadherin will be identified by immunoprecipitation and affinity chromatography with T-cadherin antibodies. These molecules will be characterized and their interaction with T-cadherin will be determined. This work will contribute to understanding how cell adhesion molecules control the generation of specific tissues in developing embryos and sustain their function in the adult. In the long term, these studies may be applied to prevent birth defects and control malignant neoplasms.
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