# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 2 Ras Dimers as New Signaling Entities and Potential Therapeutic Targets
Artificial dimerization of the KRas G12D mutant expressed at low levels (initially as membrane-bound monomers) induced strong Raf-MAPK activation and was sufficient to rescue cells from long-term serum starvation or Raf kinase inhibitor treatment [81]. Certain mutations at putative dimer interfaces (to be discussed in the next section) were shown to reduce or eliminate Raf-MAPK activation in cells [65; 97]. In addition, small peptides such as the NS1 monobody [98], designed ankyrin repeat proteins (DARPins) [99], or a natural inhibitor of Ras named DIRAS family GTPase 3 (DiRASS) (or ARH) [100] that bind Ras at a putative dimer interface and presumably block Ras dimerization also inhibit Raf-MAPK and in some cases PI3K-Akt signaling [98]. Furthermore, Ras dimerization may mediate the formation of Ras nanoclusters, which were considered to be important for effector recruitment and activation [79; 87]. The realization that Ras may function as dimers has profound implications in both basic and translational Ras biology. For an important signaling molecule like Ras, the use of dimers and higher multimers for signaling may yield performance advantages such as resistance to spontaneous fluctuations in Ras-GTP levels [101]. A number of studies have suggested a potential, tumor-suppressor role of WT Ras in mutant Ras-driven tumors [65; 102; 103; 104; 105; 106]. However, WT Ras does not compete with mutant Ras for effector binding and should not interfere with oncogenic signaling of the latter. This may now be explained with a model where Ras signaling requires two Ras-GTP molecules, and therefore heterotypic Ras-GTP:Ras-GTP dimers are incapable of driving oncogenes [33; 64]. Consistent with this explanation, development of mutant Ras-driven tumors is often accompanied with a loss of heterozygosity [103; 107; 108]. In these cases, loss of the WT _RAS_ allele and protein expression would eliminate nonfunctional Ras-GDP:Ras-GTP heterodimers and promote functional Ras-GTP:Ras-GTP homodimers, thereby enhancing mutant Ras-driven oncogenesis. As mutant Ras has been difficult to drug with conventional approaches, for example by restoring its GTPase activity or inhibiting its membrane localization, it may now be druggable by disrupting the dimers. On this front, aforementioned studies already demonstrated the potential utility of 'Ras dimer inhibitors' in targeting mutant Ras. The NS1 monobody, for example, binds both GTP- and GDP-loaded states of Ras and was shown to disrupt Ras dimerization, leading to decreased oncogenic signaling and transformation [98]. This effect was confirmed in vivo [109; 110]. Interestingly, NS1 only inhibits KRas and HRas dimerization but not NRas due to a small difference between the isoforms in the putative dimer interface. The other peptides, DARPins [99] and DiRASS [100], may also be useful in therapeutics. While both GDP- and GTP-bound Ras seem capable of dimerization, some studies suggest that GTP-binding enhances dimerization [89; 91]. If validated in vivo, this would lend a tool for selectively targeting GTP-bound Ras by inhibiting dimerization.