SNARE-mediated control of integrin trafficking, cell-ECM adhesion and focal adhesion signaling

Cell-extracellular matrix interactions are critical to cell survival and homeostasis. Disruption of extracellular matrix (ECM) interactions and the associated integrin-dependent signalling are hallmarks of many disease states, including the progression and metastatic spread of cancer. Intracellular membrane traffic is an essential component of cell-ECM interactions and recent studies suggest that SNAREs (s&barbelow;oluble N&barbelow;SF [N&barbelow; -ethylmaleimide-s&barbelow;ensitive f&barbelow;usion protein] a&barbelow;ttachment protein receptor) have important roles in cell adhesion to ECM. The goals of this research were to identify SNAREs involved in cell-ECM adhesion, and to examine the effects of inhibiting SNARE-mediated traffic on integrin signaling. It was found that a subset of endocytic-recycling SNAREs, including SNAP (synaptosomal associated protein)-23, syntaxin13 and cellubrevin/VAMP (vesicle associated membrane proteins)-3, form a complex and were required for the delivery of integrins to the cell surface, cell migration, cell spreading and the integrity of the recycling endosome. SNAP23 was also required for efficient formation of F-actin-rich membrane ruffles (induced by PMA [phorbol-12-myristate-13-acetate]); however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with the involvement of the biosynthetic-secretory pathway in membrane ruffling, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.;To assess the involvement of SNARE-mediated traffic in integrin signaling, traffic was inhibited by expression of dominant-negative (E329Q)-NSF in Chinese hamster ovary cells. E329Q-NSF expression inhibited trafficking, localization and activation of Src during spreading on fibronection, leading to decreased focal adhesion kinase (FAK)-Src interaction, and inhibited Src-dependent phosphorylation of FAK. Decreased FAK-Src interactions and activity coincided with reduced Rae activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-triphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/SrclPI3K dependent pathway.