RND1 is an atypical RHO GTPase from the RND subfamily. RND1 is constitutively bound to GTP and lacks GTPase activity. No guanine nucleotide exchange factors (GEFs), GTPase activator proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs) act on RND1. RND1 localizes to the plasma membrane, but can be extracted from the plasma membrane and sequestered to the cytosol upon phosphorylation-induced binding to 14-3-3 protein. RND1 antagonizes RHOA, leading to reduced actomyosin contractility and loss of stress fibers and focal adhesions, which results in a rounded cell phenotype. RND1 plays a role in embryogenesis, neuronal development, myometrium changes during pregnancy, and angiogenesis. RND1 is frequently downregulated in cancer and is implicated as a tumor suppressor, but may play an oncogenic role in some cancer types. RND1 expression increases in response to anti-cancer agents and in may be involved in resistance to treatment. For review, please refer to Mouly et al. 2019.

RND2 (RHON) is an atypical RHO GTPase and the least studied member of the RND subfamily. RND2 is constitutively bound to GTP and lacks GTPase activity. No guanine nucleotide exchange factors (GEFs), GTPase activator proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs) act on RND2. RND2 is predominantly expressed in brain, testis and liver (Nobes et al. 1998; Nishi et al. 1999). RND2 regulates neurite outgrowth and branching (Fujita et al. 2002; Kakimoto et al. 2004; Tanaka et al. 2006; Wakita et al. 2011) and migration of newborn neurons within the embryonic cerebral cortex (Nakamura et al. 2006; Heng et al. 2008; Alfano et al. 2011; Pacary et al. 2011; Gladwyn-Ng et al. 2015; Heng et al. 2015).

RHO family of GTPases is large and diverse, with many of its members considered to be master regulators of actin cytoskeleton. RHO GTPases are involved in the regulation of many cellular processes that depend on dynamic reorganization of the cytoskeleton, including cell migration, cell adhesion, cell division, establishment of cellular polarity and intracellular transport. As a consequence, RHO GTPases play important roles in neuronal development, immunity and cardio-vascular homeostasis. RHO GTPases are involved in the etiology of infectious diseases, congenital immunodeficiencies, neurodegenerative diseases and cancer. For review, please refer to Jaffe and Hall 2005, Lemichez and Aktories 2013, Ridley 2015, Hodge and Ridley 2016, Haga and Ridley 2016, Olson 2018, and Kalpachidou et al. 2019.

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 (Boureux et al. 2007). 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 (1) which allows them to associate with the plasma membrane (2), where they are activated by GEFs (3), and can signal to effector proteins (3.5). Then, GAPs inactivate the GTPases by accelerating the intrinsic GTPase activity, leading to the GDP bound form (4). Once again, the GDI molecules stabilize the inactive GDP bound form in the cytoplasm, waiting for further instructions (5) (Tcherkezian and Lamarche-Vane, 2007). 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). 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 (Lessey et al. 2012). RHOB and RHOC functions resemble RHOA (Vega and Ridley 2018; Guan et al. 2018). RHOB is also involved in membrane trafficking and DNA repair (Vega and Ridley 2018). RAC1 regulates the cytoskeleton and the production of reactive oxygen species (ROS) (Acevedo and Gonzalez-Billault 2018), and is involved in cell adhesion and cell migration (Marei and Malliri 2017). RAC2 expression is restricted to hematopoietic cells and RAC2 is a component of the phagocytic oxidase complex in neutrophils (Troeger and Williams 2013). 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 (Egorov and Polishchuk 2017; Xiao et al. 2018; Pichaud et al. 2019; Woods and Lew 2019). RHOJ is highly expressed in endothelial cells, regulating their motility and vascular morphogenesis (Leszczynska et al. 2011; Shi et al. 2016). RHOQ is highly activated on exocytosing vesicles and recycling endosomes (Donnelly et al. 2014) and is involved in trafficking of CFTR (cystic fibrosis transmembrane conductance regulator) (Cheng et al. 2005). RHOD regulates cytoskeletal dynamics and intracellular transport of vesicles (Randazzo 2003; Gad and Aspenstrom 2010; Aspenstrom et al. 2014). RHOF regulates cytoskeletal dynamics (Gad and Aspenstrom 2010; Aspenstrom et al. 2014) and promotes the formation of filopodia and stress fibers (Fan and Mellor 2012). 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 (Haga and Ridley 2016). RND1, RND2 and RND3 regulate cell migration (Ridley 2015; Mouly et al. 2019). RHOBTB1 is a component of a signaling cascade that regulates vascular function and blood pressure (Ji and Rivero 2016). RHOBTB2 is involved in COP9 signalosome-regulated and CUL3-dependent protein ubiquitination (Berthold et al. 2008; Ji and Rivero 2016). RHOH expression is restricted to hematopoietic cells, and it is known to be involved in T cell receptor (TCR) signaling and T cell development (Suzuki and Oda 2008; Troeger and Williams 2013). 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 (Amin et al. 2016) and 3 Rho GDIs (Dransart et al. 2005) 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”.

Repulsive Sema4D-Plexin-B1 signaling involves four GTPases, Rnd1, R-Ras, Rho and Rac1. Sema4D-Plexin-B1 binding promotes Rnd1-dependent activation of the plexin-B1 GAP domain and transient suppression of R-Ras activity. R-Ras inactivation promotes PI3K and Akt inactivation followed by GSK-3beta activation and CRMP2 inactivation. Plexin-B1 also transiently associates with and activates p190Rho-GAP, triggering a transient decrease in activated Rho.

RND3 (RHOE) is an atypical RHO GTPase from the RND subfamily. RND3 is constitutively bound to GTP and lacks GTPase activity. No guanine nucleotide exchange factors (GEFs), GTPase activator proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs) act on RND3. RND3 is a direct antagonist of ROCK1 kinase activity. RND3 prevents phosphorylation of ROCK1 targets and, similar to RND1, induces stress fiber disassembly. RND3 regulates cell migration, establishment of neuronal polarity, heart development, and myometrium changes during pregnancy. Defective RND3 function is related to cardiomyopathy, hydrocephalus and cancer. Like RND1, RND3 is implicated both as a tumor suppressor and an oncogene in cancer, and can both increase and decrease sensitivity to chemotherapeutic agents, which depends on cancer type and stage. For review, please refer to Jie et al. 2015 and Paysan et al. 2016.

RND1 binds GTP with approximately 100 times higher affinity than GDP and is thus considered to be constitutively active (Nobes et al. 1998).

Active GTP-bound RND1 binds the following effectors:
ARHGAP5 (Wennerberg et al. 2003; Bagci et al. 2020)
FRS2 (Harada et al. 2005)
FRS3 (Harada et al. 2005)
GRB7 (Vayssiere et al. 2000)
PLEKHG5 (Goh and Manser 2010)
PLXNA1 (Zanata et al. 2002)
STIP1 (de Souza et al. 2014)
STMN2 (Li et al. 2009)
UBXN11 (Katoh et al. 2002)

The following candidate RND1 effectors were reported in the high throughput screen by Bagci et al. 2020 or have been reported as RND1 effectors in some but not all studies:
ALDH3A2 (Bagci et al. 2020)
ANKRD26 (Bagci et al. 2020)
ARHGAP35 (Wennerberg et al. 2003, Mori et al. 2009: binding to active RND1; Bagci et al. 2020: no binding to active RND1)
CAV1 (Bagci et al. 2020)
CCDC88A (Bagci et al. 2020)
CPD (Bagci et al. 2020)
DEPDC1B (Bagci et al. 2020)
DLG5 (Bagci et al. 2020)
DSP (Bagci et al. 2020)
DST (Bagci et al. 2020)
EPHA2 (Bagci et al. 2020)
EPSTI1 (Bagci et al. 2020)
FAM135A (Bagci et al. 2020)
FAM83B (Bagci et al. 2020)
FLOT2 (Bagci et al. 2020)
KIDINS220 (Bagci et al. 2020)
KIF14 (Bagci et al. 2020)
LEMD3 (Bagci et al. 2020)
MUC13 (Bagci et al. 2020)
PKP4 (Bagci et al. 2020)
PIK3R1 (Bagci et al. 2020)
PIK3R2 (Bagci et al. 2020)
PTPN13 (Bagci et al. 2020)
RASAL2 (Bagci et al. 2020)
RBMX (Bagci et al. 2020)
RRAS2 (Bagci et al. 2020)
TFRC (Bagci et al. 2020)
TMEM59 (Bagci et al. 2020)
TXNL1 (Bagci et al. 2020)
VANGL1 (Bagci et al. 2020)
VANGL2 (Bagci et al. 2020)
WDR6 (Bagci et al. 2020)

RND1 does not interact with the following putative effectors that bind to active RND2 and/or RND3:
CKAP4 (Bagci et al. 2020)
CKB (Bagci et al. 2020)
DDX4 (Bagci et al. 2020)
DSG1 (Bagci et al. 2020)
FNBP1 (Bagci et al. 2020)
GOLGA3 (Bagci et al. 2020)
KTN1 (Bagci et al. 2020)
LRRC1 (Bagci et al. 2020)
NISCH (Bagci et al. 2020)
NUDC (Bagci et al. 2020)
PICALM (Bagci et al. 2020)
SCRIB (Bagci et al. 2020)
SEMA4F (Bagci et al. 2020)
TMOD3 (Bagci et al. 2020)
UHRF1BP1L (Bagci et al. 2020)

Binding of Sema3A to the Neuropilin-1-Plexin-A receptor complex results in the recruitment of Rnd1 to the cytoplasmic linker region of Plexin-A. Rnd1 activates the cytoplasmic GTPase signaling domain of Plexin-A.

Rnd1 is constitutively active and stably associates with Plexin-B1 and regulates the R-Ras GAP activity of the C1 and C2 domains of the Plexin-B1 cytoplasmic tail. These domains interact with each other and in this closed conformation cannot associate with active R-Ras-GTP. Rnd1 binds to the region between C1 and C2 domains and disrupts this interaction, allowing the receptor to associate with GTP-bound R-Ras.

Active GTP bound RND3 binds the following effectors:
ARHGAP5 (Wennerberg et al. 2003; Bagci et al. 2020)
KCTD13 (Gladwyn Ng et al. 2016)
PLEKHG5 (Goh and Manser 2010)
ROCK1 (Riento et al. 2003) RND3 inhibits kinase activity of ROCK1
TNFAIP1 (Gladwyn Ng et al. 2016)
UBXN11 (Katoh et al. 2002)

The following candidate RND3 effectors were reported in the high throughput screen by Bagci et al. 2020 or were reported by some but not all studies:
ANKRD26 (Bagci et al. 2020)
ARHGAP21 (Bagci et al. 2020)
ARHGAP35 (Wennerberg et al. 2003: binding to active RND3; Bagci et al. 2020: no binding to active RND3)
CAV1 (Bagci et al. 2020)
CCDC88A (Bagci et al. 2020)
CKAP4 (Bagci et al. 2020)
CKB (Bagci et al. 2020)
CPD (Bagci et al. 2020)
DDX4 (Bagci et al. 2020)
DEPDC1B (Bagci et al. 2020)
DLG5 (Bagci et al. 2020)
DSG1 (Bagci et al. 2020)
DSP (Bagci et al. 2020)
DST (Bagci et al. 2020)
EPHA2 (Bagci et al. 2020)
FAM83B (Bagci et al. 2020)
FLOT2 (Bagci et al. 2020)
KTN1 (Bagci et al. 2020) - RND3 has not been shown to localize to the endoplasmic reticulum membrane, but some isoforms of KTN1 have been shown to localize to the plasma membrane (Santama et al. 2004)
LEMD3 (Bagci et al. 2020)
MUC13 (Bagci et al. 2020)
NISCH (Bagci et al. 2020)
PICALM (Bagci et al. 2020)
PIK3R1 (Bagci et al. 2020)
PIK3R2 (Bagci et al. 2020)
PKP4 (Bagci et al. 2020)
PTPN13 (Bagci et al. 2020)
RASAL2 (Bagci et al. 2020)
RBMX (Bagci et al. 2020)
SCRIB (Bagci et al. 2020)
SEMA4F (Bagci et al. 2020)
TMOD3 (Bagci et al. 2020)
TXNL1 (Bagci et al. 2020)
VANGL1 (Bagci et al. 2020)
VANGL2 (Bagci et al. 2020)
WDR6 (Bagci et al. 2020)

RND3 does not interact with the following effectors:
ALDH3A2 (Bagci et al. 2020)
EPSTI1 (Bagci et al. 2020)
FAM135A (Bagci et al. 2020)
FNBP1 (Bagci et al. 2020)
FRS2 (Harada et al. 2005)
FRS3 (Harada et al. 2003)
GOLGA3 (Bagci et al. 2020)
KIDINS220 (Bagci et al. 2020)
KIF14 (Bagci et al. 2020)
LRRC1 (Bagci et al. 2020)
NUDC (Bagci et al. 2020)
ROCK2 (Riento et al. 2003)
RRAS2 (Bagci et al. 2020)
STMN2 (Li et al. 2009)
TFRC (Bagci et al. 2020)
TMEM59 (Bagci et al. 2020)
UHRF1BP1L (Bagci et al. 2020)

Rho-related GTP-binding protein Rho6