RHO GTPase cycle

Phylogenetically, RHO GTPases can be grouped into four clusters. The first cluster consists of three subfamilies: Rho, RhoD/RhoF and Rnd. The second cluster consists of three subfamilies: Rac, Cdc42 and RhoU/RhoV. The third cluster consists of the RhoH subfamily. The fourth cluster consists of the RhoBTB subfamily. Miro GTPases and RHOBTB3 ATPase are sometimes described as Rho family members, but they are phylogenetically distant from the Rho family and constitute two separate families of Ras-like GTPases, which, besides Rho, Miro and RHOBTB3 also includes Ran, Arf, Rab and Ras families. Based on their activation type, RHO GTPases can be divided into classical (typical) and atypical (reviewed by Haga and Ridley 2016, and Kalpachidou et al. 2019). Classical RHO GTPases cycle between active GTP-bound states and inactive GDP-bound states through steps that are tightly controlled by members of three classes of proteins: (1) guanine nucleotide dissociation inhibitors or GDIs, which maintain Rho proteins in an inactive state in the cytoplasm, (2) guanine nucleotide exchange factors or GEFs, which destabilize the interaction between Rho proteins and their bound nucleotide, the net result of which is the exchange of bound GDP for the more abundant GTP, and (3) GTPase activating proteins or GAPs, which stimulate the low intrinsic GTP hydrolysis activity of Rho family members, thus promoting their inactivation. GDIs, GEFs, and GAPs are themselves subject to tight regulation, and the overall level of Rho activity reflects the balance of their activities. Many of the Rho-specific GEFs, GAPs, and GDIs act on multiple Rho GTPases, so that regulation of these control proteins can have complex effects on the functions of multiple Rho GTPases (reviewed by Van Aelst and D'Souza-Schorey 1997, and Hodge and Ridley 2016). The classical Rho GTPase cycle is diagrammed in the figure below. External or internal cues promote the release of Rho GTPases from the GDI inhibitory complexes, which allows them to associate with the plasma membrane, where they are activated by GEFs (1), and can signal to effector proteins (4). Then, GAPs inactivate the GTPases by accelerating the intrinsic GTPase activity, leading to the GDP bound form (2). Once again, the GDI molecules stabilize the inactive GDP bound form in the cytoplasm, waiting for further instructions (3). Classical RHO GTPases include four subfamilies: Rho (includes RHOA, RHOB and RHOC), Rac (includes RAC1, RAC2, RAC3 and RHOG), Cdc42 (includes CDC42, RHOJ and RHOQ) and RhoD/RhoF (includes RHOD and RHOF) (reviewed in Haga and Ridley 2016). RHOA, the founding member of the RHO GTPase family, regulates the actin cytoskeleton, formation of stress fibers and cell contractility, which is implicated in cell adhesion and migration. RHOB and RHOC functions resemble RHOA. RHOB is also involved in membrane trafficking and DNA repair. RAC1 regulates the cytoskeleton and the production of reactive oxygen species (ROS), and is involved in cell adhesion and cell migration. RAC2 expression is restricted to hematopoietic cells and RAC2 is a component of the phagocytic oxidase complex in neutrophils. RAC3 shares 92% sequence identity with RAC1 and is highly expressed in neurons (de Curtis 2019). CDC42 regulate the cytoskeleton and cell polarity, and is involved in cell adhesion and migration as well as in intracellular membrane trafficking. RHOJ is highly expressed in endothelial cells, regulating their motility and vascular morphogenesis. RHOQ (also known as TC10) is highly activated on exocytosing vesicles and recycling endosomes and is involved in trafficking of CFTR (cystic fibrosis transmembrane conductance regulator). RHOD regulates cytoskeletal dynamics and intracellular transport of vesicles. RHOF regulates cytoskeletal dynamics and promotes the formation of filopodia and stress fibers. RHOD and RHOF do possess GTPase activity and are therefore grouped with classical RHO GTPases, but they are atypical in the sense that they possess high intrinsic guanine nucleotide exchange activity and do not require GEFs for activation.

Atypical RHO GTPases do not possess GTPase activity. They therefore constitutively exist in the active GTP-bound state. Atypical RHO GTPases include three subfamilies: Rnd (includes RND1, RND2 and RND3), RhoBTB (includes RHOBTB1 and RHOBTB2), RhoH (RHOH is the only member) and RhoU/RhoV (includes RHOU and RHOV). RND1 and RND3 can antagonize RHOA activity, leading to loss of stress fibers and cell rounding. RND1, RND2 and RND3 regulate cell migration. RHOBTB1 is a component of a signaling cascade that regulates vascular function and blood pressure. RHOBTB2 is involved in COP9 signalosome-regulated and CUL3-dependent protein ubiquitination. RHOH expression is restricted to hematopoietic cells, and it is known to be involved in T cell receptor (TCR) signaling and T cell development. RHOU and RHOV expression is induced by WNT signaling and they are involved in regulation of cell shape and cell adhesion (Faure and Fort 2015; and Hodge and Ridley 2016).

Almost every classical RHO GTPase interacts with multiple GEFs, GAPs and GDIs, and every RHO GTPase activates multiple downstream effectors. There are 82 Rho GEFs (71 Dbl, reviewed in Fort and Blangy 2017, and 11 DOCK, reviewed in Meller et al. 2005), 66 Rho GAPs and 3 Rho GDIs encoded by the human genome. To keep our reaction annotations compact, we have grouped the GEF, GAP, GDI and effector proteins associated with each RHO GTPase into sets. Within a set, we have distinguished full set members from candidate members on the basis of the amount of experimental evidence supporting the member’s molecular function. Note that members of a set can otherwise be functionally quite diverse. Annotation of upstream activators of GEFs, GAPs and GDIs was outside the scope of this catalogue pathway and is or will be shown elsewhere in Reactome. Signaling through downstream effectors is shown in more detail in the Reactome pathway “RHO GTPase Effectors”.