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Reconstitution Of Rho Gtpase Interactions At The Membrane
In order for fibroblasts to migrate, budding yeast to polarize, and macrophages to undergo phagocytosis, each cell must coordinate a membrane-localized signal with a robust morphological change. The Rho Family of GTPases play critically important roles in mediating these cellular processes. However, in order for the Rho GTPases to help regulate these processes in a tightly coordinated spatial and temporal manner, they need to be properly localized to specific membrane signaling sites. The mechanisms by which this precise localization is achieved are still not fully understood, but represent the subject of this thesis. The Rho GTPases are post-translationally modified at their C-terminus by the covalent attachment of a 20 carbon lipid tail, called a geranylgeranyl moiety, which allows them to associate with membranes. To facilitate their cytosolic localization, the Rho GTPases require the assistance of a ubiquitously-expressed regulatory protein, the Rho guanine nucleotide-dissociation inhibitor (RhoGDI). The mechanism by which the Rho GTPases move between distinct locations in the cell, and in particular, how they are able to cycle on and off between different membranes, has been a challenging question. I have set out to begin to define this mechanism by reconstituting in vitro the interactions between the geranylgeranylated Rho GTPase Cdc42, RhoGDI, and liposomes of well-defined lipid composition. In taking advantage of the sensitivity and real-time capabilities of these reconstituted systems, some unexpected findings emerged regarding how RhoGDI influences the membraneto-cytosol distribution of Rho GTPases like Cdc42. One such unexpected discovery involves my finding that RhoGDI can distinguish between the signaling-inactive (GDP-bound) and signaling active (GTPbound) forms of Cdc42 when they are associated with a lipid bilayer. In particular, despite having similar affinities for the GDP- and GTP-bound forms of Cdc42 in solution, I found that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared to its GTPbound counterpart. Moreover, the membrane-release of Cdc42-RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI then being to significantly slow the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol. I further demonstrated that the cycling of Rho GTPases like Cdc42 and Rac1 between the membrane and cytosol can be strongly influenced by RhoGEFs and RhoGAPs, as well as RhoGDI, such that the membrane association-dissociation cycle of the Rho GTPases is directly coupled to their GTP-binding/GTPase cycle. Most of the Rho family GTPases contain a cluster of positive-charged residues (i.e. a 'polybasic domain'), directly preceding their geranylgeranyl moiety. It has been suggested that this domain serves to fine-tune their localization among different cellular membranes. I have used reconstituted systems to examine the role of the polybasic domain of Cdc42 in its ability to bind to membranes. These studies highlight a key role for a di-arginine motif close to the C-terminus of Cdc42 in binding to phosphatidylinositol-4,5-bisphosphate (PIP2). While this interaction does not influence the ability of activated Cdc42 to signal the necessary changes to impact the actin cytoskeletal architecture and induce filopodia/microspikes, it is essential for the ability of an oncogenic mutant of Cdc42 to transform fibroblasts. Thus, these findings highlight the importance of Cdc42 being localized to a PIP2-enriched membrane site to engage those signaling partners that mediate oncogenic transformation.
Cerione, Richard A
Collins, Ruth N.; Baird, Barbara Ann
Molecular & Cell Biology
Ph.D. of Molecular & Cell Biology
Doctor of Philosophy
dissertation or thesis