# The RHO Family GTPases: Mechanisms of Regulation and Signaling
## 2 The RHO Family and the Molecular Switch Mechanism  
Members of the RHO family have emerged as key regulatory molecules that couple changes in the extracellular environment to intracellular signal transduction pathways. To date, 20 canonical members of the RHO family have been identified in humans and can be categorized into distinct subfamilies based on their sequence homology: RHO (RHOA, RHOB, and RHOC); RAC (RAC1, RAC1B, RAC2, RAC3, and RHOG); CDC42 (CDC42, G25K, TC10, TCL, WRCH1, and WRCH2); RHOD (RHOD, RIF); RND (RND1, RND2, and RND3); and RHOH [47]. RHO family proteins are approximately 21-25 kDa in size. They typically contain a conserved GDP-/GTP-binding domain, called the G domain, and a C-terminal hypervariable region (HVR) ending with a consensus sequence known as CAAX (C is cysteine, A is any aliphatic amino acid, and X is any amino acid) (Figure 2). The G domain consists of five conserved sequence motifs (G1 to G5) that are involved in nucleotide binding and hydrolysis [48]. In the cycle between the inactive and active states, at least two regions of the protein, switch I (G2) and switch II (G3), undergo structural rearrangement and transmit an \"OFF\" to \"ON\" signal [3]. Subcellular localization, which is known to be critical for the biological activity of RHO proteins, is achieved through a series of posttranslational modifications at a cysteine residue in the CAAX motif including isopenylation (geranylgeranyl or farnesyl), endoproteolysis, and carboxyl methylation [49]. Figure 2: Domains, signature motifs, and post-translational modification of RHO GTPases. RHO GTPases contain a highly conserved G domain, which is responsible for GDP/GTP binding and GTP hydrolysis. Switch I and switch II regions are the consensus binding sites for GEFs, GAPs, GDIs, and effectors, and undergo conformational changes upon the nucleotide exchange and hydrolysis [3]. restof captionAll members of the RHO family contain conserved glycine 12 (G) and glutamine 61 (Q: RAC1 numbering), except for the RND proteins, which contain, among other deviations, other residues at these positions. This is why RND proteins constantly remain in the GTP bound state [50]. Other signatures are, for example, a 19-amino acid insertion next to the switch II region in RAC1B with drastic biochemical consequences [51], and glutamic acids (E) in CDC42 crucial for a selective WASP interaction [52]. Some members have amino acid insertion outside the G domain (yellow boxes) with yet unknown properties. The N-terminal insertion in WRCH1 contains proline-rich motifs responsible for interaction with SH3-containing adaptor proteins [53]. Most members have comparable biochemical properties such as nucleotide binding, exchange, and hydrolysis. In contrast to most members, which end up under resting conditions in an inactive GDP-bound state, RAC1B, RHOD, and RIF cumulate in the GTP-bound state due to a faster intrinsic nucleotide exchange reaction (k\({}_{\text{diss}}\)) compared to the intrinsic GTP hydrolysis reaction (k\({}_{\text{cat}}\)) [54]. The C-terminal hypervariable region (HVR) contains the terminal CAAX box, which undergoes posttranslational modification by geranylgeranylation (GG) or alternatively famesylation (F) in the case of RHOB and the RND proteins at the conserved cysteine (green). Additional modification by a palmitoyl (P) moiety has been reported for RHOB, and the CDC42-related proteins TC10, TCL, and WRCH1. These modifications lead to the membrane anchorage of the members, a process that is stabilized and potentiated through variable numbers of positively charged arginine and lysine residues (+). Once an isoprenoid moiety is added to CAAX, a RHO protein is translocated to the endoplasmic reticulum, where RCE1 cleaves the AAX tripeptide tail, and then, RHO undergoes carboxymethylylation by ICMMT [55] RHO proteins can also be phosphorylated, which can affect their association with their regulators or effectors or influence their membrane stability [56; 57; 58].