U161534.pdf (12.74 MB)
The regulation of integrin vesicular trafficking
thesisposted on 2014-12-15, 10:32 authored by Marnie. Roberts
Growth factors are able to influence cell adhesion and migration by regulating the function of integrins. Integrins engage in endo/exocytic cycling and I have investigated the possibility that growth factors influence integrin function by controlling their endocytosis and/or recycling. In serum starved mouse fibroblasts avb3 and a5b1 integrins are internalised, trafficked to the perinuclear recycling compartment, and then returned to the cell surface in a Rab11-dependent fashion. In contrast, following addition of PDGF, avb3 integrin (but not a5b1) was returned directly from the early endosomes to the plasma membrane via a pathway dependent on Rab4, and not Rab11. Moreover, growth factor regulated integrin recycling was not restricted to fibroblasts, but occurred in human endothelial cells in response to VEGF. Inhibition of avb3 recycling using dominant negative Rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for avb3), but adhesion to fibronectin (a ligand for a5b1 and avb3) was unchanged indicating that Rab4-depdnent recycling is essential for avb3 function.;PDGF and VEGF are known to activate the PI(3)K/ PKB/Akt signalling axis and recent evidence indicates that this pathway is involved in modulating integrin function. Recycling of both avb3 and a5b1 was inhibited by expression of dominant negative PKB and, furthermore, constitutively active PKB stimulated the flux of avb3 from the early endosome to the plasma membrane. Blockade of PKB/Akt by dominant negative mutants compromised cell spreading on vitronectin and fibronectin, consistent with a requirement for recycling in the function of both avb3 and a5b1 integrins.;To gain insight into the biochemical events occurring during integrin activation I looked for active signalling molecules that are recruited to avb3 following PDGF treatment. A 44kDa protein rich in phosphotyrosine and phosphothreonine coimmunoprecipitated with avb3 integrin and western blotting revealed this protein to be active pp44 ERK1.
Date of award2002-01-01
Awarding institutionUniversity of Leicester