The RHO Family GTPases: Mechanisms of Regulation and Signaling

Abstract

Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.

Keywords: CDC42; RAC1; RHO signaling; RHOA; RHOGAP; RHOGDI; RHOGEF; effectors.

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]. All 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-contining 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_dis) compared to the intrinsic GTP hydrolysis reaction (k_cat) [54]. The C-terminal hypervariable region (HVR) contains the terminal CAAX box, which undergoes posttranslational modification by geranylgeranylation (GG) or alternatively farnesylation (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 (+).

Figure 1

Molecular principles of RHO GTPase regulation and signaling. Most RHO GTPases (20 canonical human members) act as molecular switches by cycling between a GDP-bound, inactive state and a GTP-bound, active state. They interact specifically with four structurally and functionally unrelated classes of proteins: (A) In resting cells, guanine nucleotide dissociation inhibitors (GDIs; 4 human members) sequester RHO in the cytoplasm, away from the membrane, by binding to the lipid anchor and thus creating an inactivated cytosolic pool; (B) in stimulated cells, different classes of membrane receptors activate guanine nucleotide exchange factors (GEFs; 85 human members: 74 DBL and 11 DOCK family proteins), which in turn activate RHO by accelerating the intrinsic exchange of GDP for GTP and switch ON signal transduction; (C) active GTP-bound RHO interacts through the GTPase-binding domain (GBD) with and activates downstream targets (effectors; >70 human members) to cause a variety of intracellular pathways, which control a multitude of biochemical processes involved in the regulation of different biological (dys)functions; (D) GTPase-activating proteins (GAPs; 66 human members) negatively regulate RHO by stimulating its slow intrinsic GTP hydrolysis activity and switch OFF signal transduction. Notably, all RHO-interacting proteins recognize and bind RHO at consensus-binding sites called switch I and II.

Figure 3

Domain organization of DBL family proteins. The DBL family RHOGEFs are mostly multimodular proteins and have a number of functional domains that may mediate cross talk between RHO proteins and other signaling pathways. DH domains are almost always found with a PH domain in the C-terminus. Some DBL proteins contain two DH–PH cassettes, while some DBL proteins lack tandem PH domains. Functional domains, in addition to the catalytic DH domain (green), 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 do not exhibit activity under cell-free conditions [47].

Figure 4

Domain organization of the RHOGAP family proteins (adapted from Amin 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].

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.