Spatiotemporal control of Ras plasma membrane interactions

The plasma membrane is a complex, dynamic structure with heterogeneity on many length and time scales. The origins of this heterogeneity are diverse and include several thousand species of lipid, compartmentalization by a sub-membrane actin cytoskeleton and the lateral assembly of sphingolipids and cholesterol into transient liquid-ordered (Lo) domains. Plasma membrane lateral heterogeneity in turn imposes a non-random distribution on proteins across different nanoscale domains. We have studied plasma membrane lateral heterogeneity on the nanoscale in the context of Ras proteins and shown that H-, K-ras, N-ras operate in spatially discrete, transient, dynamic nanoclusters on the plasma membrane. Interestingly, H-, K- and N-ras all undergo GTP-regulated segregation between different types of nanodomain. Our results show that approximately 30-40% of Ras proteins assemble into clusters of 6-8 proteins on the plasma membrane. These protein-lipid assemblies, are small and short- lived, with a radius of ~9nm and lifetime of 0.1-0.5s. Importantly, Ras.GTP nanoclusters operate as digital switches that are used by the cell to build high-fidelity signal transduction circuits. Our current focus is to determine the structural, biophysical and molecular basis for Ras nanoclustering. To date these studies have revealed a complex interplay between the C-terminal lipid anchor, flanking C-terminal protein sequences and specific helices in the actual G-domain that regulate Ras interactions with the lipid bilayer. We have also identified a novel switch mechanism that regulates different modes of lipid bilayer attachment according to G-domain conformation. More broadly the critical role for plasma membrane lipids in the molecular mechanisms of lateral segregation suggests that lipophilic and amphiphilic drugs may be used to perturb plasma membrane nanoclustering by modifying lipid / lipid interactions. Intriguingly, we find that a wide range of such commonly prescribed drugs stabilize Lo domains in model and biological membranes and have striking effects on Ras lateral segregation. We also observe significant effects on Ras nanoclustering in cells treated with certain classes of Raf inhibitor, albeit by a completely different molecular mechanism. Taken together these results suggest that diverse mechanisms of protein nanoclustering on the plasma membrane may be novel drug targets.