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 that they act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focussed 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 both in 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 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, B and C, Rac1, RhoH and Wrch2/RhoV bound to GTP were found to decrease following co-expression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.
