# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 1 Introduction
The signaling process is terminated when the bound GTP is hydrolyzed into GDP, typically aided by GAPs since the intrinsic GTPase activity of Ras is low [35; 38]. This scheme of regulation ensures that Ras-GTP is kept at low levels in resting cells and becomes elevated only upon proper upstream stimuli (Figure 2). Mutations in the \(RAS\) genes, commonly at the G12, G13, and Q61 residues, or loss of Ras GAPs such as NF1, impair GTP hydrolysis to result in upregulated Ras-GTP, ultimately leading to oncogenic transformation and other, pathologic conditions [17; 18]. Figure 1: Ras domain structure. Left panel shows a ribbon representation of the heart-shaped Ras G-domain using overlaid KRas-GTP (Protein Data Bank (PDB) 6 GOD; GTP shown in green) and KRas-GDP (PDB 4 GBE; light pink; GDP in plum) structures as models. Several key elements most relevant to this review, including Switch I (residues 30–40, magenta), Switch II (residues 60–70, red), \(\beta\)-sheets 2 (\(\beta\)2, residues 37–46, sky blue), \(\beta\)3 (residues 49–58, sky blue), and \(\alpha\) helices 3 (\(\alpha\)3, residues 87–103, bronze), \(\alpha\)4 (residues 127–137, bronze), and \(\alpha\)5 (residues 152–164, bronze) are highlighted in the KRas-GTP model. Right panel depicts the hypervariable regions (HVRs) of the four major Ras isoforms, namely HRas, NRas, KRas (4A) and KRas (4B) along with the membrane targeting signals (famesylation, palmitoylation, and the poly-lysine stretch). Figure adapted from ref [32]. Membrane localization provides another critical layer of regulation for Ras activities in cells. Ras isoforms, notably the ubiquitous expressed H, N, and KRas (4A and 4B), are highly homologous in their G-domain sequences but differ substantially in their C-terminal 23-24 amino acids or so-called the HVRs (Figure 1, right) [39]. The HVR comprises a linker plus a membrane anchor, terminated with a CAAX (C=Cysteine, A=Aliphatic; X=Any) motif that is farnesylated post translation, followed by cleavage of the AAX [40; 41]. At this stage membrane binding by the farnesylated cysteine is weak and requires strengthening by additional mechanisms [42; 43]. NRas and KRas4A HVRs are further modified with one palmitoyl group whereas the HRas HVR takes two palmitoyl groups [44]. In contrast, KRas4B (hereafter KRas) contains a hexa-lysine polybasic sequence in its HVR that can electrostatically interact with acidic lipid headgroups to stabilize its membrane binding [45]. Aside from driving membrane localization of Ras, the modified HVRs are also responsible for the dynamic interactions of Ras with distinct membrane domains in an HVR-dependent (i.e., isoform-specific) manner [39; 46]. As such, the Ras isoforms differ in effector specificity despite nearly identical G-domains and ability to bind to the same set of effectors in vitro [39; 46; 47]. Indeed, HRas, NRas, and KRas have overlapping but nonidentical functions [39; 48; 49; 50] and distinct mutational spectra in human cancers [2; 39; 51; 52]. Thus, while the Ras G-domain is functionally capable of mediating diverse biological activities, the signaling specificity may be primarily encoded by the HVR and its associated post-translational modifications [29]. The most recent and least understood mechanism is the formation of Ras multimers, including dimers and higher order oligomers (commonly referred to as Ras nanoclusters). Ras GTPases were traditionally considered monomeric G-proteins, but high-resolution imaging studies revealed that a significant fraction of Ras exists in multimers on the cell membrane and that formation of these structures is critical to the activation of Raf-MAPK and potentially other effector pathways [32; 53]. When two Ras-GTP molecules come into contact, they could bring two effector molecules--each bound to a Ras-GTP monomer--into proximity, which may facilitate effector dimerization and activation. Indeed, at least one major Ras effector, the Raf kinase, depends on dimerization for activation [54].