# The RHO Family GTPases: Mechanisms of Regulation and Signaling
## 4 Downstream Effectors of RHO GTPases
### Structural Characteristics of RHO GTPase-effector Interactions  
The crystal structures of the GTPase-binding domains (GDDs) of PKN and RHO kinase (ROCK) in complex with RHOA revealed that the domains, as predicted from their primary structure, form \(\alpha\)-helical coiled coils that are arranged in an antiparallel and parallel fashion, respectively [143; 144]. A 13-residue left-handed coiled coil in the  
Figure 5: Regulation of actin-based motility by RHOA, RAC1, and CDC42. Activated CDC42, RAC, and RHO bind to and specifically activate their downstream effectors, which are either kinases (e.g., ROCK, PAK, and PI5K) or scaffolding proteins (e.g., DIA, WASP, IRSp53, and IQGAP). These effector proteins activate diverse signaling pathways with distinct effects on the actin cytoskeleton and cellular morphology. An important aspect of cell motility is the equilibrium between the myosin light chain (MLC) and phosphorylated MLC, which is tightly regulated. C-terminal portion of the ROCK-GBD, which is considered the minimal sequence required for RHO-interacting motif activity, binds exclusively to the switch and \(\alpha\)2 regions of RHOA. In contrast, the RHOA-PKN complex has two possible contact sites on RHOA [143]: contact site 1 consists of the \(\alpha\)1, \(\beta\)2/\(\beta\)3, and \(\alpha\)5 regions of RHOA, whereas contact site 2 overlaps remarkably well with the ROCK-binding site. The structures of CDC42 in complex with effector proteins containing a CDC42/RAC-interactive binding (CRIB) motif such as PAK1 and WASP, which have been determined mostly by NMR spectroscopy due to their high flexibility [145; 146; 147; 148; 149], have shown that the GBD in this class of effectors makes extensive contact with the surface of RHO GTPases. Specifically, GBD binds through its \(\beta\)-hairpin and C-terminal \(\alpha\)-helix to the \(\alpha\)1, switch I, and II regions and wraps around the \(\alpha\)5 and \(\beta\)2 regions of the GTPase with its extended N-terminus, which encompasses the CRIB motif. The basic region of WASP, immediately upstream of the CRIB motif, has been shown to generate favorable electrostatic steering forces to unique glutamate residues in CDC42 (Glu49, Glu171, and Glu178) that control the accelerated WASP-CDC42 association reaction (Figure 2) [52; 150]. This process is a prerequisite for WASP activation and a critical step in the temporal regulation and integration of WASP-mediated cellular responses (Figure 5). Two other effectors, arfaptin and p67\({}^{\text{phox}}\), have novel structures and contact sites on the GTPase [151; 152]. Arfaptin forms an elongated crescent-shaped dimer with three helix coiled-coils that makes contact with the switches I and II and \(\alpha\)2 regions of RAC1, regardless of its nucleotide-bound state [152], and structurally mimics the DH domain of Tiam1 [153]. p67\({}^{\text{phox}}\) has an \(\alpha\)-helical domain that consists of four tetratricopeptide repeat (TPR) motifs, which bind \(\alpha\)1, the N-terminal residues of switch I, and the G3 and G5 loops, but not the switch II region or the principal parts of switch I [151]. It has been proposed that the switch regions might be the contact sites for a third protein that is associated with the Rac1-GTP-p67\({}^{\text{phox}}\) complex [154; 155]. The mechanism of effector activation of the GTPase-effector complex structures mentioned thus far have not been clarified; however, intramolecular autoinhibition and exposure of their functional domains are known to be required. A common feature of effector complexes is that, with the exception of p67\({}^{\text{phox}}\), they all make intensive contact with the switch/\(\alpha\)2 regions of RHO GTPases, which indicates that this region probably serves as the platform for the GTP-dependent recognition of effectors. Two invariant leucine residues (Leu69 and Leu72), which form crucial hydrophobic contacts with almost all effector domains, have been proposed as essential elements for the CDC42/RAC-mediated activation of CRIB-containing effectors [148].