Regulation of [beta]1 integrin function by aberrant N-linked glycosylation and Vacuolar H[superscript plus]-ATPase 16-kDa subunit expression
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Integrins are a family of cell surface glycoproteins that functions as cell adhesion receptors to the extracellular matrix (ECM). The interactions between integrins and ECM play important roles in regulating cell adhesion, migration, and proliferation. In particular, β1 integrin dimerizes with multiple numbers of integrin α subunits, leading to the most diverse array of interactions between the cells and ECM. This dissertation research studied how the function of β1 integrin is affected by vacuolar H+-ATPase 16-kDa subunit expression. It was previously shown that overexpression of vacuolar H+-ATPase 16 kDa (16K) subunit inhibited β(1,6) branched N-glycans of β1 integrin, thus blocking β1 integrin-dependent cell migration toward laminin and fibronectin. Expression of 16K inhibited β1 integrin-calnexin association in the endoplasmic reticulum, thereby disrupting the folding and subunit assembly of β1 integrin for functional α/β1 integrin expression on the cell surface. This inhibition resulted in Triton X-100 insoluble, non-disulfide-linked aggregation of minimally glycosylated β1 integrin in the endoplasmic reticulum. During the course of this study, we also found that calnexin associates with 16K transiently in the endoplasmic reticulum for 16K biosynthesis. Our results provide a new insight into how 16K-β1 integrin interaction potentially leads to down-regulation of β1 integrin surface expression and subsequent β1 integrin-mediated signal transduction, which is highly implicated in resistance of certain tumor cells to cancer chemotherapy. Another focus of this dissertation was to study effect of aberrant glycosylation on neuronal differentiation. N-acetylglucosaminyltransferase VB (GnT-VB) is a Golgi glycosyltransferase that is selectively expressed in brain and neural retina of developing mouse embryo. Expression of GnT-VB in rat pheochromocytoma PC12 cells was used as a model system to investigate if GnT-VB has a significant role in neuromorphogenesis. The results showed that GnT-VB expression promotes an increased rate of neurite outgrowth on collagen and laminin substrates in a β1 integrin-dependent manner. Using neurite outgrowth by manganese-induced β1 integrin activation and β1 integrin function-blocking antibody, it was shown that GnT-VB enhanced neurite outgrowth is most likely mediated by alteration of β1 integrin-ECM interaction. These results suggested that N-linked glycosylation modification of cell surface glycoproteins by GnT-VB may function during neuronal differentiation.