# The RHO Family GTPases: Mechanisms of Regulation and Signaling
## 3 Regulation of RHO Family GTPases
### GTPase-Activating Proteins (GAPs)  
Hydrolysis of bound GTP is the timing mechanism that terminates signal transduction of RHO family proteins and enables their return to an inactive, GDP-bound state [87]. The intrinsic GTPase reaction is usually slow but can be stimulated to accelerate by several orders of magnitude through interaction with RHO-specific GAPs [112; 113]. The RHOGAP family is identified by the presence of a conserved catalytic GAP domain that is sufficient for engaging RHO proteins and mediating accelerated catalysis [114; 115]. The GAP domain inserts a conserved arginine residue, termed an \"arginine finger\", into the GTP-binding site of the cognate RHO protein to stabilize the transition state and catalyze the GTPase reaction [74; 76; 77]. This mechanism is similar to that of other small GTP-binding proteins including RAS, RAB, and ARF, although the sequence and folding of the respective GAP families differ from other GTP-binding proteins [115; 116]. Masking the catalytic arginine finger is an elegant mechanism for the inhibition of GAP activity. This action has also been recently discovered in the tumor suppressor protein DLC1, an RHOGAP, which is competitively and selectively inhibited by the SH3 domain in p120 RASGAP [117; 118].  
The first RHOGAP discovered, p50 RHOGAP, was identified through a biochemical analysis of human spleen cell extracts in the presence of recombinant RHOA [119]. The majority of RHOGAP family members typically harbor several other functional domains and motifs that are implicated in tight regulation and membrane targeting (Figure 4) [74; 82; 83]. Numerous mechanisms have been shown to affect the specificity and catalytic activity of RHOGAPs (e.g., intramolecular autoinhibition [120], posttranslational modification [121], and regulation by interaction with lipid membranes [122] and proteins [118]).  
RHOGAP insensitivity has been frequently analyzed through the substitution of either amino acid that is critical for GTP hydrolysis by RHO proteins (e.g., Gly14 or Gln63 in RHOA), and these mutations generate proteins known as constitutively active mutants [123; 124]. In other mutants, the catalytic arginine residue of the GAP domain is replaced with an alanine residue [113; 124]. The latter approach is, in principle, very useful under cell-free conditions but not optimal in cells because an Arg-to-Ala mutant may provide a readout similar to that of the wild-type protein as it interferes with downstream signaling by competing with effector(s) for binding to RHO proteins. These RHOGAP mutants are able to bind persistently to their target protein, sequestering the target, which most likely leads to a readout similar to that of activated wild-type RHOGAP. Therefore, it has recently been suggested that mutating critical \"binding determinants\", particularly Lys319 and Arg323 (p50 numbering), may be a better strategy than substituting the catalytic arginine [114]. Charge reversal of these residues most likely leads to loss of RHOGAP association with its substrate RHO protein and thus abrogates the activity of the GAP domain. This outcome renders mutagenesis not only a tool for determining the specificity of RHOGAPs, but also for investigating GAP domain-independent function(s) of the RHOGAPs.  
Figure 4: Domain organization of the RHOGAP family proteins (adapted from [PERSON] et al., 2016 [114]). RHOGAPs are mostly multimodular proteins and have a number of functional domains that may mediate cross talk between RHO proteins and other signaling pathways. Functional domains, in addition to the catalytic GAP domain (red), are probably involved in lipid and membrane binding (blue), protein interactions (bright green), and enzymatic activities (red and orange). A scale of amino acid numbers in increments of 200 is shown at the bottom. Underlined proteins are GAP-like proteins with no RHOGAP activity [114].