Rho GDIs are the third class of regulators for Rho GTPases. They interact with the small GTPase in a 1:1 stoichiometry complex and block nucleotide exchange on the small GTPase 106, 107. Mammals have three GDIs: the ubiquitously expressed RhoGDI (GDI1), the hematopoietically restricted GDI2 (LyD4GDI) and RhoGDI3 (also called RhoGDIγ) 108. GDI1 and 2 are cytosolic, whereas GDI3 is membrane bound and it is normally found on intracellular vesicles 109. The crystal structure of RhoGDI in complex with Cdc42 has been resolved, leading to a deeper understanding on the mechanism of action of GDIs 110. RhoGDI is composed of an N terminal arm that binds to the switch I and II of the small GTPase, blocking nucleotides exchange. The C terminal part of RhoGDI has an immunoglobulin shape and if forms a hydrophilic pocket that is able to host the prenyl motif of the small GTPase, thereby stabilizing the Rho protein in the cytosol 110. Interestingly, RhoGDI is equally affine for Cdc42-GDP and Cdc42-GTP in the cytosol. However, on membranes, its affinity towards the small GTPase changes: GDI1 is now more affine to Cdc42-GDP 107. X-rays crystal structure did not show appreciable differences in the position of the switch I and II of soluble Cdc42 bound to GDP or GTP 111. It is therefore possible to speculate that the association with membranes may help GTP-bound Cdc42 to assume a conformational state that is more readily distinguishable from the GDP-bound GTPase and, thus, more receptive to bind target/effector proteins. RhoGDI does not actively extract Cdc42 from membranes. In fact, Johnson et al. showed that in vitro Cdc42 can dissociate from lipids at the same rates with or without GDI 107. Rather than directly promoting dissociation from membranes, GDI1 stabilizes Cdc42 as it spontaneously dissociates from membranes. Given that GDI1 binds preferentially to Cdc42-GDP on membranes, the overall
Introduction – Small Rho GTPases - 48
result will be the accumulation of GTP on membranes and the stabilization of Cdc42-GDP in the cytosol 107. The same mechanism works also for Rac1, but not for RhoA 107.
Figure 2 - Model representing the mechanism of action of GDIs. GDIs bind to Cdc42-GDP on membranes, stabilizing it as soon as it spontaneously leaves the membrane. Therefore, GDI slows down the reassociation of Cdc42-GDP to membranes. In the cytosol, GDIs can interact with the same affinity with both GTP and GDP bound Cdc42. Cdc42-GTP is more affine for membranes than Cdc42-GDP, therefore the net result of all these movements will be the enrichment of Cdc42-GTP on membranes
There is evidence for GDIs playing important roles in the modulation of intracellular signaling.
Overexpression of GDI1 results in decreased levels of active Cdc42, which in turn inhibit RasGRF capacity to activate Ras. Therefore, GDI1 overexpression results in a reduction of MAPK signaling 105. However, this observation should be interpreted carefully as GDI bound Cdc42 is not able to interact with RasGRF or with any other GEF, thus it is difficult to understand how the complex GDI-Cdc42 could modulate RasGRF with respect to its activity toward Ras. An alternative explaination would be that binding of Cdc42 to RasGRF positively modulates Ras activation and overexpression of GDIs would remove Cdc42 from RasGRF, thus reducing its GEF activity towards Ras. Binding of GDI to Cdc42 is important for the ability of Cdc42 to induce transformation 112. A mutant of Cdc42 that undergoes faster GDP/GTP exchange (Cdc42-F28L) was shown to promote transformation in NIH3T3 cells 113. When a mutation that disrupts the binding between Cdc42 and GDI (R66A) was introduced into Cdc42-F28L, the ability of Cdc42-F28L-R66A to induce transformation was completely lost 112. Several important questions remain to be answered to fully understand the role of GDIs. First, how are GDIs dissociated from the small GTPase, in order to reactivate signaling downstream
Introduction – Small Rho GTPases - 49
of the Rho proteins? Second, are GDIs simply cytosolic anchors, or could they work as chaperons for the small GTPases?
To answer the first question, a possibility is that another class of proteins will lead to rapid dissociation of the GDI from the small GTPase. This is the case for Rab GTPases, where GDF (GDI Dissociation Factors) displace the RabGDI from Rabs, thereby making it possible for a RabGEF to activate the small GTPase 114. However, to date, no evidence of such proteins has been reported for Rho GTPases. Another possibility is that GDIs are phosphorylated and phosphorylation makes them less affine for the small GTPases. For instance, PAK1 phosphorylates GDI1 on Ser101 and Ser174, leading to decrease in its affinity for Rac 115. In addition, Src can phosphorylate GDI1 on Tyr156 in vitro and in cells. Phosphorylated GDI1 translocates to membranes and shows reduced affinity towards Rho GTPases 116. Finally, aPKC has been reported to phosphorylate RhoGDI, resulting in this case in the liberation of Rac1
117. Alternatively to GDIs being phosphorylated, there is evidence that phosphorylation of small GTPases can change their affinity for GDIs 118. Forget et al. showed that PKA phosphorylation of Cdc42 and RhoA increases the affinity of those two small GTPases towards GDI1 118.
GDIs can bind also to GTP loaded GTPases, the binding preventing interaction with effectors of the Rho proteins and with GAPs 107. Once the small GTPase is GDI bound, it is protected by GAPs, meaning that it cannot be inactivated, unless before dissociation of GDI is stimulated.
This raises the possibility that GDIs can act as chaperones to translocate active small GTPases between different subcellular locations 119, 120. In agreement with this idea, it was observed that the Cdc42 mutant incapable of binding GDI1 was localized more prominently at the Golgi than the GDI bound protein 112. The same different localization upon impaired binding to GDI1 was described also for Rac1 121. In another study from Boulter et al, depletion of GDI1 resulted in reduced plasma membrane localization of Cdc42, Rac1 and RhoA and concomitant increase of their localization on endomembranes 122. Boulter et al. observed also that in mammalian cells, the level of RhoGDI1 is roughly equivalent to the total levels of RhoA, Rac1 and Cdc42 combined. Therefore, Rho GTPases compete for the binding to GDI and any condition that alters the level of one GTPase will result in disruption of this balance, affecting the normal on/off cycle of Rho GTPases 122.
One last function of GDIs is to prevent degradation of small GTPases. Once the Rho protein dissociate from membranes, their geranylgeranilation causes them to unfold and therefore
Introduction – Small Rho GTPases - 50
they are degraded by the proteasome 122. By stabilizing the prenylation, GDIs prevent degradation of small GTPases. This allows cells to have a constant pool of small GTPases that can be rapidly activated in response to stimuli.
Introduction – Small Rho GTPases - 51
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