# Ras Isoforms from Lab Benches to Lives--What Are We Missing and How Far Are We?
## 5 Missing Pieces in the Ras Puzzle
### Microdomain Localization of Ras Isoforms  
The prime platform of Ras signaling is the inner leaflet of the plasma membrane. The three isoforms localize to the cholesterol-rich-detergent-resistant raft, as well as non-raft depending on their state of activation. H-Ras is tethered to the plasma membrane by its HVR comprising of two palmitoyl residues. Studies using fluorescence-based vectors for H-Ras have shown that the targeting motif interacts with the cholesterol-rich domain. Another crucial motif in H-Ras's interaction with the plasma membrane is the linker region that connects the effector domain to the HVR region [127]. On GTP loading, this linker induces a conformational change in H-Ras, laterally segregating it from a cholesterol-dependent to cholesterol-independent domain. Of the dual palmitoyl residues, Cysteine 184 is sufficient for the GTP binding dependent lateral segregation of H-Ras between the two domains. Contrary to this, if singly palmitoylated on cysteine 181, lateral segregation of H-Ras on GTP binding occurs from cholesterol-independent to cholesterol-dependent domains [128; 129]. Galectin-1 acts as a scaffold protein to further stabilize the interaction of active H-Ras with the cholesterol-independent microdomain [130]. A few studies have pointed out that H-Ras itself might be important for raft formation, as it induces negative changes in the curvature of the plasma membrane [131]. This leads to the recruitment of more lipids into this area of curvature and thus a lipid-dense region is formed. Contrary to H-Ras, N-Ras undergo lateral segregation into cholesterol-dependent nanoclusters upon GTP-binding. In both H- and N-Ras, the lateral segregation is also dependent on the spacing between the palmitoyl group and the prenyl group [128; 129]. K-Ras has a farnesylated cysteine and a polylysine residue for its interaction with the membrane. The important property of the polylysine residue is its negative charge which enables membrane tethering [132]. K-Ras resides in cholesterol-independent microdomains enriched with lipids like phosphatidylserine and phosphatidyl inositol 4,5-bisphosphate. No study has yet elucidated GTP-dependent lateral segregation of K-Ras. However, galectin 3 binds to K-Ras-GTP and stabilizes it [133]. Latrunculin, an inhibitor of actin cytoskeleton completely abolishes the formation of cholesterol-dependent nanoclusters at the inner leaflet demonstrating the importance of intact actin cytoskeleton in this process [128; 129].  
[PERSON] et al. have also discussed in their study the relative size difference of Ras isoform nanoclusters in the inner leaflet of the plasma membrane. Using immunogold labeling of the plasma membrane microdomains, they found that the mean number of gold particles in the H-Ras G12V cluster was 2.5 that corresponds to about 6 H-Ras proteins, whereas for K-Ras G12V clusters it was 3.25, corresponding to roughly 7.7 K-Ras proteins. This difference in the size of the nanoclusters may translate into differences in the mechanism of nanocluster formation of the two isoforms [129].  
A recent study showed the spatiotemporal preference of Ras isoforms at the plasma membrane [134]. Phosphatidylserine (PtdSer) affords K-Ras a capability to mechanosense membrane curvature and oncogenic K-RasG12V mutant prefers a flatter plasma membrane with low curvature. Corroborating with it, the K-Ras-dependent MAPK pathways are more active in flatter cells with low plasma membrane curvature [134]. Contrarily, dually palmitoylated H-Ras favor a curved membrane as also demonstrated by the finding that H-Ras-PI3K signaling is more stimulated in cells with a curved surface. Prostate cells expressing active H-Ras have an elongated morphology when compared to cells expressing H-Ras dominant-negative mutant H-Ras17N which have a flat morphology [135; 136].