Background
Rho family GTPases are members of the Ras GTPase superfamily and act as molecular switches in numerous signaling pathways that control a variety of cellular processes, including actin cytoskeletal organization, microtubule dynamics, vesicle trafficking, cell cycle progression, and cell polarization [1]. Most Rho GTPases cycle between active GTP-bound and inactive GDP-bound states. There are three classes of regulators of Rho GTPases, namely, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and GDP-dissociation inhibitors (GDIs).
GEFs activate GTPases by promoting the exchange of GDP for GTP. GAPs inactivate GTPases by stimulating their intrinsic GTP-hydrolyzing activity. GDIs are known to regulate only members of the Rho and Rab families and not other families of the Ras superfamily, although a GDI-like protein for Ras GTPases has been reported [2]. Unlike GEFs and GAPs, GDIs play several roles in the regulation of the Rho family GTPases [3-6]. First, GDIs bind GDP-bound GTPases and inhibit the dissociation of GDP from GTPases, thereby preventing the promotion of GDP/GTP exchange by GEFs and maintaining the GTPases in an inactive state [7]. Second, although the binding affinity of GDIs to GTP-bound GTPases remains controversial [8-15], it is possible that GDIs bind GTP-bound GTPases and inhibit both intrinsic and GAP-promoted GTP hydrolyzing activity [8,16,17], thereby maintaining GTPases in an active state. Third, GDIs mediate the cycling of GTPases between cytosolic and target sites [7].
GDIs for the Rho family GTPases can therefore act to inhibit both the activation and inactivation of GTPases by interacting with GDP- and GTP-bound GTPases, respectively. This dual function of GDIs is noteworthy, and adds to our understanding of the regulatory mechanisms of the Rho GTPase cycle, because GDIs for Rab family GTPases show a marked preference for the GDP-bound form [18]. Furthermore, it has also been suggested that Rho GTPases are regulated by a fine balance between GEF and GAP activities, and that the inactivation of GAP activity is a physiologically important regulatory mechanism for activating Rho GTPases [19]. Nonetheless, little is known about the significance of the inhibition of GAP-promoted GTP hydrolyzing activity by GDIs in the regulation of Rho signaling. How the opposing roles of GDIs influence the Rho GTPase cycle is also unclear.
Several ordinary differential equation models and process models of the Rho GTPase cycle have been constructed and analyzed [20-24]. In these models, GDIs inhibit the functions of GEFs and GAPs by sequestering GDP-bound and GTP-bound GTPases, respectively. However, the actual mechanisms involved in GDI inhibition of GEF and GAP activity are not fully understood. A previous report suggested that RhoGDIs can physically interact directly with both GEFs [25] and GAPs [26]. Based on these observations, we constructed a model of the Rho GTPase cycle in which GDIs inhibit the activities of GEFs and GAPs not only by sequestering GTPases, but also by direct physical interaction.
Using this model, we showed that the functions of GEFs and GAPs are integrated into Rho GTPase signaling through the interactions of these regulators with GDIs and that the negative role of GDIs is to suppress the overall Rho activity by inhibiting GEFs. Additionally, the positive role of GDIs is to sustain Rho activation by inhibiting GAPs. These observations illustrate the more detailed roles RhoGDIs and further enhance our understanding of the physiological functions of Rho GTPase signaling.