Chen, Yun-Ju
(2003)
The role of dystroglycan in cell adhesion.
PhD thesis, University of Glasgow.
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Abstract
Dystroglycan is a heterodimeric transmembrane glycoprotein protein of α and β subunits that links the extracellular matrix to the cytoskeleton. Recent data tend to suggest that the role of dystroglycan in non-muscle cells is for cell adhesion, and cytoskeleton reorganisation signalling. To study the relationship between dystroglycan signalling and ERK signalling in focal adhesions, an YFP-ERK construct was expressed in REF52 cells. YFP-ERK was expressed with ERK activity and formed adhesion-like structures in the REF52 cells, however, no ERK activity was detected in the adhesion-like structures in the REF52 cells. To determine the function of dystroglycan for adhesion or cytoskeleton organisation in non-muscle cells, a GFP-tagged full-length dystroglycan (αβDG-GFP) or its deletion mutants was expressed in REF52 cells. Expression of αβDG-GFP markedly altered cell phenotype on a laminin or fibronectin substrate, resulting in the induction of actin-rich filopodia. The β-dystroglycan cytoplasmic domain is determined as the mediator for the dystroglycan-dependent filopodia formation mediated partly by integrin signalling. The dystroglycan deletion mutants lacking α-dystroglycan failed to target to the plasma membrane. Expression of an alkaline phosphate-tagged β-dystroglycan cytoplasmic domain construct (AP-cβ), which targeted the β-dystroglycan cytoplasmic domain to the membrane without the α-dystroglycan, was sufficient to induce the filopodia phenotype, indicating that with the proper membrane localisation, β-dystroglycan can regulate filopodia formation independently of α-dystroglycan. However, α-dystroglycan might be necessary for β-dystroglycan to target to the plasma membrane. These distinct morphologies strongly implied that β-dystroglycan mediates Cdc42 regulated cytoskeleton reorganisation. By cotransfecting dominant negative Cdc42 (Cdc42N17) or constitutively activated Cdc42 (V12Cdc42) constructs with αβDG-GFP or the mutants, Cdc42 was determined to be a mediator for dystroglycan-dependent filopodia formation. Therefore a signalling cycle of dystroglycan-Cdc42-PAK-ezrin-dystroglycan is identified. With this signalling cycle, dystroglycan plays a role in inducing actin filopodia formation and, at the same time, might also inhibit focal adhesion and stress fibre formation.
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