Integrin-mediated adhesion regulates the transmission of many signals downstream from growth factor and cytokine receptors that regulate cell proliferation, migration, differentiation and survival. Many of these signaling events depend on cholesterol-enriched membrane microdomains (or CEMMs) that are enriched in lipid-modified proteins and sphingolipids. Our previous work showed that in normal, anchorage-dependent cell types, the small GTPase Rac, a critical mediator of cell motility and proliferation, can be biochemically activated independent of adhesion but its targeting to the plasma membrane and interaction with effectors still require integrin engagement. Rac membrane binding sites are within CEMMs and integrins control their localization such that detachment of cells from the substratum triggers rapid clearance of CEMMs from the plasma membrane and movement to an intracellular compartment. Replating cells triggers exocytosis of CEMMs to restore plasma membrane localization. Internalization of CEMMs is mediated by caveolae and requires phosphorylation of a small fraction of caveolin-1 on tyrosine 14. Conversely, exocytosis of CEMMs during replating requires the small GTPases Ral A. Based on this preliminary and published data, we propose to: 1. Investigate the mechanism of Rac activation and membrane targeting to determine whether CEMMs cooperate with guanine nucleotide exchange factors, what is the role of RhoGDI in Rac activation, and whether in vivo dynamics requires """"""""displacement factors"""""""" that accelerate dissociation of Rac and RhoGDI. 2. Investigate the role of RalA in CEMM exocytosis. We will further test the involvement of Ral in integrin- stimulated exocytosis, investigate pathways downstream of Ral and mechanisms of Ral activation. 3. Investigate mechanisms by which phospho-Y14 caveolin triggers caveolar endocytosis. Proteins that interact with caveolin or caveolar membranes will be identified and studied. The requirement for dynamin in endocytosis will also be investigated. ? ? ?
Showing the most recent 10 out of 48 publications
