# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 4 Membrane Interactions Regulate Ras Enrichment, Orientation, and Dimerization  
Ras proteins are now known to interact with membrane lipids via both the modified HVRs and their G-domains. Previous studies have established the roles of these interactions in the membrane binding of Ras and its enrichment in nanoscopic spatial domains in an isoform and nucleotide dependent manner. Recent work has started to suggest that these interactions also play a role in determining the orientation of Ras on the membrane with direct bearings on how Ras dimerizes. Most studies to date suggest a weak interaction between the Ras G-domains [89; 91; 92; 115], therefore additional factors are likely needed to promote the formation of dimers and higher multimers. One such factor is to reduce the dimensionality of Ras diffusion, from a 3D solution phase to a 2D plane via membrane binding, and further, from a 2D plane to \(\sim\)1D nanoscopic domains. This reduction in dimensionality is primarily achieved through interactions between the lipidated Ras HVR and the membrane with contributions from the G-domain [126; 127]. Once on the membrane, the distinct HVRs and the associated lipidation patterns of each Ras isoform direct Ras to distinct, nanoscopic domains, leading to enrichment of Ras in highly confined spaces [78; 79; 80; 128; 129]. This targeting is achieved dynamically by Ras sampling the membrane (Figure 4) [130; 131; 132]. In the case of KRas, for example, the molecule diffuses on the membrane freely with fast mobility (diffusion coefficient or D \(\sim\)1 \(\upmu\)m\({}^{2}\)/s) until it enters domains that are \(\sim\)200 nm in size and confer intermediate mobility (D \(\sim\) 0.3 \(\upmu\)m\({}^{2}\)/s). Within the latter domain reside much smaller structures (\(\sim\)70 nm or smaller in diameter) that ultimately confine and immobilize KRas (D \(<\) 0.1 \(\upmu\)m\({}^{2}\)/s), where KRas is internalized [130] and presumably forms dimers or nanoclusters during its residence [133]. Other Ras isoforms appear similar in their apparent diffusion and confinement behavior [134; 135], although the underlying interactions are isoform-specific [132; 136]. Computer simulations and cell biology studies suggest that the HVR-mediated spatial segregation of Ras is in large part driven by the interactions between its post-translational modifications, notably palmitoylation and prenylation, and membrane lipids. Whereas the fully saturated palmitoyl group prefers an ordered membrane domain such as the lipid rafts,the unsaturated farnesyl group (with 'kinks' in the aliphatic chain) prefers a less ordered domain [137]. The KRas HVR and similar polybasic peptides can bind to PS through both electrostatic interactions and specific molecular recognition [138; 139]. Aside from the HVR, the Ras G-domain also makes contacts with the membrane and contributes to the spatial segregation and confinement, which could account for the spatial segregation of GTP- vs GDP-bound forms of Ras [140; 141]. GDP-bound HRas and GTP-bound NRas incorporate cholesterol into their respective nanoclusters whereas GTP-bound HRas and GDP-bound NRas form cholesterol-independent nanoclusters [72]. Cholesterol was proposed to be important for de-mixing between different types of Ras nanoclusters [142]. KRas preferentially binds lipid bilayers containing PIP2 in part driven by its G-domain interactions with the anionic lipid [126; 143; 144]. Together, these mechanisms direct specific Ras forms (isoform and/or nucleotide-bound form) to distinctive, confined spaces where Ras-Ras interactions can be facilitated. Once in the nanodomains, the dimerization dynamics will largely be dictated by what is surrounding Ras. Dimerization requires the two Ras protomers to be properly oriented relative to each other, thus limiting the conformations compatible with dimer formation. Interactions between Ras and anionic lipids such as PS and PIP2 in its immediate  
Figure 4: Ras diffusion on the membrane and localization to nanodomains.