Analysis of cadherin and receptor tyrosine kinase interactions: trafficking and function.

Bryant, David Michael (2006). Analysis of cadherin and receptor tyrosine kinase interactions: trafficking and function. PhD Thesis, Institute for Molecular Bioscience , University of Queensland.

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Author Bryant, David Michael
Thesis Title Analysis of cadherin and receptor tyrosine kinase interactions: trafficking and function.
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Associate Professor Jennifer Snow
Abstract/Summary In multicellular organisms, sheets of polarized epithelial cells provide a functional barrier between the external world, and the internal milieu. One protein critical for epithelial polarization and cell-cell adhesion is the epithelial cadherin, or E-cadherin protein. Although the adhesive function of E-cadherin is mediated primarily at the cell surface, E-cadherin undergoes complex internalisation and intracellular trafficking in response various different physiological circumstances. The mechanisms that regulate these pathways remain poorly defined. A central aim of this thesis has been to characterise the pathways and machinery that regulate internalisation of E-cadherin. To this end, mammary adenocarcinoma (MCF-7) cells were examined for E-cadherin trafficking, cell-cell adhesion and morphogenetic status in response to stimulation with various growth factors, primarily fibroblast growth factors (FGFs), which play a key role in mammary development and tumourigenesis. FGF stimulation induced the cointernalisation of E-cadherin and the FGF receptor 1 (FGFR1) into early endosomes, resulting in disrupted cell-cell adhesion. FGFR1 translocated to the nucleus in response to a number of stimuli. These studies reveal endocytosis as an initial, requisite step for the subsequent nuclear translocation of FGFR1 by a still uncharacterised pathway. In a novel finding, the E-cadherin-catenin complex was found to conversely regulate signalling and nuclear translocation of FGFR1 in response to ligand. The internalisation pathway for E-cadherin was found to be independent of caveolin-1, yet only displayed partial dependence on classical clathrin-based endocytic pathways, suggesting the potential of either an unusual endocytic pathway or of multiple internalisation pathways. Interestingly, stimulation of the same cell type with an alternate growth factor, epidermal growth factor (EGF) resulted in internalisation of free E-cadherin through macropinocytosis, without disruption to polarized adhesion. Surprisingly, neither of these pathways resulted in a significant reduction in E-cadherin protein levels. Additional analyses of cellular machinery revealed recruitment of members of the sorting nexin (SNX) protein family to E-cadherin-containing vesicles, whereby SNX1 was noted to regulate intracellular sorting of E-cadherin away from the degradative pathway. Analysis of the morphogenetic effect of cadherin internalisation revealed that whilst a number of stimuli were able to induce internalisation of E-cadherin, stimulation with FGF alone was able to induce scattering and migratory activity in MCF-7 cells. Curiously, this behaviour was independent of E-cadherin expression, revealing a novel partial epithelial-mesenchymal transition (pEMT) in response to FGF. These findings underscore the notion that E-cadherin is able to utilise multiple pathways for internalisation, depending on growth factors, and on cellular context. Finally, studies using epithelial cell lines embedded in extracellular matrix to form glandular like structures suggest the potential for intracellular trafficking of E-cadherin to be studied in 3-D using epithelial cell culture models. These studies reveal the existence of alternate pathways for E-cadherin internalisation and provide an important insight in the differential regulation of E-cadherin localization and function under different physiological circumstances.

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Created: Fri, 21 Nov 2008, 14:58:33 EST