# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 2 Ras Dimers as New Signaling Entities and Potential Therapeutic Targets  
Until the last \(\sim\)5 years, the dominant view of Ras GTPases had been that they are monomeric G-proteins, often compared with the family of trimeric G-proteins. This notion was affirmed with the observations that soluble Ras proteins containing only the G-domain are able to bind effectors in solution, and that multimers observed in crystals of Ras proteins seemed to be packing artefacts. The use of new imaging tools, including immunoelectron microscopy (EM) [73] and then super resolution microscopy [74; 75; 76; 77], has allowed visualization of Ras proteins on the cell membrane with nanometer resolution. With these tools, it has been shown repeatedly that formation of Ras multimers on biological membranes is common. Immuno-EM imaging of prepared membrane sheets showed that \(\sim\)40% of all Ras proteins exist in multimers, each consisting of 5-8 Ras molecules, and that the Ras multimers are spatially segregated depending on the isoform and nucleotide bound [78; 79; 80]. Super resolution imaging revealed that 10-20% of Ras molecules are present in dimers when expressed at near-endogenous levels, and the fraction of higher order multimers only becomes evident at higher expression levels [81]. Aside from nanoscopic imaging, Ras dimers have been probed with a multitude of other experimental approaches. In their original paper in 2000, [PERSON] et al. observed KRas-GTP dimerization on liposomes after exposure to a chemical crosslinker, which requires a lipidated HVR, and HRas G12V (GTP) dimerization in cells by means of \(\beta\)-galactosidase bimolecular complementation [82]. Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy and foster resonance energy transfer (FRET) [83] were used to detect NRas dimers on artificial membranes [84]. Single-molecule FRET was recently used to probe NRas dimers and measure the distance between specific sites on the two Ras protomers [85]. FRET and the related fluorescence life-time imaging microscopy (FLIM) [86] were used in many studies to detect proximities between Ras molecules in cells [65; 67; 84; 87]. Negative-stain EM with structural fitting revealed an interesting, trimeric organization of KRas on a lipid monolayer [88]. Mass-spectrometry in combination with ultraviolet photodissociation was used to compare the dimerization tendencies of various G12X variants [89]. Nuclear Magnetic Resonance (NMR) has been a powerful tool to study transient and weak interactions [90] such as Ras dimerization in solution [91]. Most recently, NMR with paramagnetic relaxation enhancement (PRE) was used to study KRas dimerization on nanodiscs [71; 92]. Importantly, several studies also reported the absence of Ras dimerization. On supported lipid bilayers, the [PERSON] lab combined single-particle tracking, fluorescence correlation spectroscopy, and step-wise fluorescence bleaching to study potential homogenization of HRas [93] and KRas [94]. Within a wide range of protein densities and lipid compositions, the authors did not find indications of dimer formation with either Ras isoform. The originally observed HRas dimers were later found to be artefacts due to laser-induced crosslinking of fluorophores [95]. Using a different set of approaches including time-domain fluorescence anisotropy and NMR, [PERSON] et al. also did not observe any G-domain interactions even when two HRas molecules were linked in tandem using flexible linkers of various lengths [96]. Rather than proving that Ras dimers do not exist, these studies suggest that biologically meaningful Ras-Ras interactions may only take place under certain conditions that, at present, are best afforded by biological membranes. There has been increasing evidence in support of a critical role of Ras dimers in effector activation, at least in the case of Raf-MAPK signaling.