A Complete Survey of RhoGDI Targets Reveals Novel Interactions with Atypical Small GTPases

Abstract

There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the “typical” or “classical” small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with “atypical” small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.

Post-Translational Modification and Subcellular Compartmentalization: Emerging Concepts on the Regulation and Physiopathological Relevance of RhoGTPases

“…RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases protecting them from degradation. A recent systematic study shows that the three different RhoGDIs (1, 2 and 3) interact with all possible Rho family small GTPases raising the possibility of a differential functional modulation depending on the RhoGDI binding isoform [21]. Mechanisms other than GTP-GDP cycling and GDI binding also modulate Rho GTPase signaling, including transcriptional and post-transcriptional expression regulation [22,23] (Figure 1).…”

Pathophysiological Mechanisms in Neurodevelopmental Disorders Caused by Rac GTPases Dysregulation: What’s behind Neuro-RACopathies

“…Twenty small Rho family guanosine triphosphatases (GTPases) have been identified in humans, classified in eight major subfamilies based on structural and biological properties: Rho-, Rac-, Cdc42-, RhoU/RhoV-, Rnd-, RhoD/R-, hoF-, RhoBTB-, and RhoH subfamilies [ 1 , 2 ]. As relevant key regulators of cytoskeletal dynamics and intracellular signaling, Rho family guanosine triphosphatases (GTPases) are crucial for the regulation of several fundamental biological processes in various cell types, including cell cycle progression, gene transcription, and cell morphology, motility, and polarity [ 3 ].…”

“…While GEFs promote the dissociation of GDP and the binding of GTP, resulting in the activated state of the protein, GAPs stimulate the intrinsic GTPase activity and the GTP hydrolysis, thus terminating the signaling and completing the cycle [ 13 , 14 ]. Acting as negative regulators, GDIs are instead essential to regulate the amount of available GTPases and also modulate their targeting to intracellular compartments [ 1 , 2 ]. Once in the active state, the GTPases can activate several distinct downstream effectors, ranging from actin-related proteins to kinases ( Figure 2 ) [ 15 ].…”

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells

“…The vast majority is maintained in the cytosol through GDIs ( Garcia-Mata et al, 2011 ). As opposed to GEFs and GAPs, GDIs exist in a limited number with only three members (RhoGDI-1, -2, -3, RabGDIα, -β, -3) identified so far ( Nazlamova et al, 2017 ; Muller and Goody, 2018 ; Ahmad Mokhtar et al, 2021 ).…”