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Regulation of the RhoGTPases by RhoGDI

and .

Signal transduction pathways mediated by the Rho-family GTPases require tight temporal and spatial control. The GDI proteins are key components of the regulatory machinery controlling both the timing and the localization of Rho GTPase activity. Recent structural work has provided significant insight into the mechanisms by which the GDI proteins control Rho-family signalling. In this review, the basis of the three distinct biochemical activities of the GDI toward the Rho GTPases, namely (1) inhibition of nucleotide dissociation, (2) inhibition of GTP hydrolysis, and (3) membrane release, are described in the context of this structural information. Understanding the biochemistry of the GDI provides a starting point for exploring the cell biology of this important class of regulatory molecules and recent progress in understanding the roll of the GDI in the cell is also discussed.

Introduction

Members of the Rho family of GTPases are remarkable in their ability to regulate an enormous range of cellular responses (described in reviews of this series and in references 1, 2). The ability to bind and hydrolyze GTP lies at the heart of the biological activity of the Rho GTPases, allowing them to function as molecular switches cycling between an active, GTP-bound conformation and an inactive, GDP-bound conformation. In the GTP-bound state, Rho GTPases are able to specifically couple to downstream effector proteins and activate their biological activities, giving rise to signals that control important cellular processes including organization of the actin cytoskeleton, maintenance of cell polarity, lipid signalling, membrane trafficking, gene expression, and oncogenic transformation. Given the complex signalling networks governed by Rho-family proteins, it is clear that both the timing and the localization of the GTP binding and hydrolytic cycle must be tightly controlled. To this end, the cell has evolved a series of regulatory factors responsible for controlling signalling events mediated by the Rho GTPases. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) catalyze activation via GDP/GTP exchange and inactivation via GTP hydrolysis, respectively (reviewed in this series and in refs. 3-5). Guanine nucleotide dissociation inhibitors (GDIs) represent a third class of regulatory proteins that are critical to the control of signalling events mediated by the Rho GTPases. The GDIs are unique among regulatory proteins in that they exhibit multiple effects on their Rho-family substrates, controlling both the nucleotide state of the GTP-binding proteins as well as their cellular location. Recently, significant progress has been made toward understanding the structural basis of the interaction of the Rho GTPases with GDIs. As described in the following review, this structural information provides important insights into the signalling events mediated by the Rho GTPases and provides a context for understanding the cellular function of the GDIs.

GDIs As Multifunctional Regulators of Signalling through Rho GTPases

The GDI proteins comprise a family of regulatory factors that control signalling by the Rho proteins (previously reviewed in ref. 6) and exhibit three distinct biochemical activities toward these GTPases (Fig. 1). The GDI proteins were initially identified based on their ability to bind to the inactive, GDP-bound form of Rho proteins and block nucleotide dissociation.7-9 In this capacity, the GDI acts as a negative regulator of Rho-family signalling, blocking GEF-mediated exchange and maintaining the GTPase in an inactive GDP-bound state. Subsequently, it was shown that RhoGDI is also able to bind with high affinity to the GTP-bound form of the Rho family member Cdc42 to act as a GTPase inhibitory protein (GIP),10 blocking both intrinsic and GAP-catalyzed GTP-hydrolysis and thus maintaining Cdc42 in a GTP-bound conformation. The third, and perhaps the most important cellular activity of the GDI, is its ability to solubilize Cdc42 from cellular membranes.11,12

Figure 1. Biochemical activities of the GDI.

Figure 1

Biochemical activities of the GDI. The GDI serves as a multi-functional regulator of signalling by Rho GTPases. The three distinct biochemical activities of the GDI (1) inhibition of nucleotide dissociation, (2) inhibition of GTP hydrolysis, and (3) membrane (more...)

In the absence of a GDI, Rho proteins are stably associated with cellular membranes by virtue of a series of post-translational modifications directed toward their carboxy-terminal CAAX motif13 (see also Chapter 2). The most significant of these modifications is the covalent attachment of a prenyl moiety to the CAAX box cysteine. In most cases this lipid group is a 20-carbon geranylgeranyl group attached by geranylgeranyl transferase I14 (RhoB, RhoD and RhoE are modified with the smaller 15-carbon farnesyl group). Following prenylation, a specific protease cleaves the last three residues of the CAAX motif, and the new carboxyl-terminal prenylated cysteine is carboxy-methylated. The hydrophobic prenyl moiety, along with a polybasic region proximal to the modified cysteine, directs the Rho family proteins to specific membrane-bound locations where they carry out their cellular function.15 RhoGDI binds only to the prenylated form of Rho proteins, and GDI binding sequesters the lipid moiety from the membrane, creating a soluble cytosolic GDI/GTPase complex. Appropriate localization of Rho proteins is critical for their biological function, and regulation of this localization by RhoGDI is likely to play an important role in signalling through Rho GTPases (see also Chapter 2).

A GDI activity acting on Rho and Cdc42 was independently purified from bovine brain cytosol by two groups.7-9 The sequencing and cloning of the protein responsible for this activity lead to the identification of RhoGDI (also known as GDI1 or αGDI ), the first Rho family specific GDI protein. In addition to RhoGDI, two related proteins have been described (Fig. 2). The Ly- or D4-GDI (also referred to as GDI2 and β GDI) was found as a GDI protein preferentially expressed in hematopoietic cells16-18 with 74% sequence identity to RhoGDI. In spite of the high degree of conservation at the level of primary sequence, GDI2 interacts with a 20-fold lower affinity toward Cdc42. This decreased binding affinity has been attributed to a single amino-acid change from Ile177 in RhoGDI to an asparagine at the corresponding position in GDI2.19 Two groups have recently described a third member of the GDI-family, referred to as GDI3 (also called γGDI).20,21 This protein is enriched in brain and appears to have a high degree of specificity to RhoB and RhoG and also binds more weakly to Cdc42 and RhoA but not Rac. GDI3 has a unique, extended amino-terminal region comprised of an amphipathic α-helix. Unlike the other members of this family, which are entirely cytosolic, GDI3 is associated with a Triton-X insoluble fraction and appears to localize to vesicular structures in the vicinity of the ER. While the conserved nature of the primary sequence of GDI3 suggests a common mode of interaction with Rho-family substrates, the distinctive noncytosolic localization and substrate specificity point to a specialized role, possibly in the regulation of RhoB signalling.6

Figure 2. The RhoGDI family.

Figure 2

The RhoGDI family. Three Rho family-specific GDI proteins have been identified, each exhibiting a high degree of primary sequence conservation (indicated as percent identity). The domain architecture of the GDIs described in the text is illustrated in (more...)

Structural Insights into GDI Function

We have recently described the structure of the Cdc42/RhoGDI complex22 which, along with the structure of the Rac1/GDI2 complex,23 the recently described Rac2/RhoGDI structure,24,25 and a low resolution structure of the RhoA/RhoGDI complex,26 provides a consistent mechanistic picture of the regulation of Rho GTPases by the GDIs. In addition, these structures reveal important differences between the RhoGDI and GDI2 proteins and provide important insights into the cellular function of these regulatory proteins. The structure of the post-translationally modified form of Cdc42 bound to RhoGDI is shown in (Fig. 3). The molecular surface of the GDI is drawn to emphasize the domain architecture of the GDI, which is comprised of two distinct domains, a well-ordered carboxy-terminal domain and a more flexible amino-terminal region.

Figure 3. Structure of the GTPase/GDI complex.

Figure 3

Structure of the GTPase/GDI complex. (A) A ribbon diagram of the Cdc42/GDI complex is shown with Cdc42 in yellow, with the switch I in red and switch II in orange, and GDI in blue. The Mg2+ ion and GDP are shown in the nucleotide-binding pocket of Cdc42 (more...)

Earlier structural work on the carboxy-terminal domain of the GDI27,28 showed that this region adopts an immunoglobulin-like fold, comprised of two anti-parallel β-sheets that pack against one another to form a β-sandwich. The most striking feature of the GDI immunoglobulin-like domain is the presence of a unique hydrophobic cavity between the two sheets of the βsandwich, which provides a binding site for the geranylgeranyl moiety. In the absence of Cdc42 binding, the hydrophobic pocket of the GDI is too small to accommodate the lipid. Upon complex formation, the base of the pocket dramatically expands to allow insertion of the geranylgeranyl group. In the structure of the Cdc42/GDI complex, the geranylgeranyl moiety of Cdc42 is completely sequestered from the solvent, and the binding of the lipid group within this hydrophobic cavity provides a mechanism for release of the GTPase from cellular membranes.

In contrast, the geranylgeranyl moiety was not visible in the electron density map of the Rac1/GDI2 structure.23 The lack of an ordered geranylgeranyl group in the Rac1/GDI2 complex reflects a weak affinity of the GDI2 for binding to the lipid moiety. As noted above, a single amino acid change from isoleucine at position 177 of RhoGDI to an asparagine at the corresponding position in GDI2 accounts for a 20-fold difference in binding affinity for Cdc42.19 Isoleucine 177 in RhoGDI forms hydrophobic contacts with the geranylgeranyl group important in stabilizing the lipid in the binding pocket. The introduction of a polar asparagine residue at this position is likely to disrupt geranylgeranyl binding as seen by the disordered nature of the lipid in the Rac1/GDI2 complex. Importantly, the structure of Rac2 bound to RhoGDI24 clearly shows the presence of the geranyl-geranyl group in the hydrophobic pocket with a conformation nearly identical to that seen for the Cdc42/RhoGDI complex. This structure confirms that lack of a well ordered geranylgeranyl moiety in the Rac2/GDI2 structure is due to the differences in lipid binding between the two GDI proteins, rather than differences in the GTPases, and may reflect functional differences between these GDIs.

In each of these structures, the amino-terminal region of the GDI folds into a well-ordered helix-loop-helix, and this "regulatory-arm" of the GDI interacts with the switch I and switch II regions of the Rho protein to influence the nucleotide state of the GTPase. Importantly, NMR spectroscopy of the full-length GDI shows that the amino-terminal region of the GDI is disordered in the absence of Cdc42 binding.27-29 As predicted by these NMR studies, the structure of the complex clearly shows that this flexible region adopts an ordered structure upon binding to the GTPase. The amino-terminal region of the GDI is essential for its function, and biochemical studies show that while deletion of the first 22 residues from the GDI has only slight affects on its interaction with Rho proteins, deletions of 45 or 60 residues completely abrogates binding and blocks GDI activity.30 The amino-terminal region of the GDI contributes a significant portion of the protein-protein interactions at the complex interface, and the structural data demonstrates that this region is responsible for blocking guanine nucleotide dissociation and inhibition of GTP hydrolysis (described in detail below).

A number of recent NMR studies have investigated the functional importance of the disordered amino-terminal region of the GDI. These studies show that the free GDI is in equilibrium between two conformations, one in which the amino-terminus is in a random coil and a second in which the amino-terminal domain transiently adopts a helical structure similar to that seen in complex with Rho GTPases.29,31 Mutations in the amino-terminal domain that favor the random coil structure dramatically interfere with GDI binding to Rac1.32 The structure of the Cdc42/GDI complex shows that the extreme amino-terminal region of RhoGDI (residues 10-15) forms a short helical region that caps the geranylgeranyl binding pocket, further sequestering the lipid moiety from the solvent. In the Rac1/GDI2 structure this region is disordered, consistent with NMR data showing that this region of RhoGDI exhibits transient helical structure while GDI2 does not. These differences in the helical nature of the amino-terminal region may also contribute to the disparity in lipid binding and the divergent function of these two GDIs.

As emphasized by these NMR studies, a critical aspect of complex formation is stabilization of the flexible amino-terminal region of the GDI. A universally conserved arginine residue in switch II (Arg66 in Cdc42) is crucial to the GTPase/GDI interaction and is involved in a series of interactions that buttress the flexible regulatory arm of the GDI against the stable immunoglobulin like domain.22 In the absence of these bridging interactions, the amino-terminal region of the GDI will not adopt the stable fold required for interacting with the switch regions of the GTPase. Based on the central importance of the conserved arginine in switch II, we predicted from the structural data that mutation of this residue would render Rho GTPases unable to interact with the GDI. The importance of this arginine in the Cdc42/GDI interaction was recently demonstrated biochemically by Gibson and Wilson-Delfosse, showing that the Cdc42(R66A) mutant is unable to couple to the GDI and, thus, localizes exclusively to the membrane fraction.33 Importantly, this mutation appears to be specific to the GDI interaction, as other known effectors and regulatory factors do not rely on this arginine residue for binding. As described in the following sections, the structural data provides a mechanistic understanding of the three biochemical functions of the GDI; however, the cellular role of these regulatory interactions remains less clear. GDI-insensitive mutants, such as the Cdc42(R66A) mentioned above, will be a valuable reagent in unraveling the role of the GDI in signalling events mediated by the Rho GTPases.

Mechanism of the GDI Activity

A primary function of the GDI is to block the dissociation of the bound nucleotide and inhibit the exchange activity of Dbl-family GEFs. Recently the structure of Rac in complex with its specific GEF Tiam1 was determined,34 and comparison of this structure with that of the Cdc42/GDI complex provides important insight into the antagonistic activities of these two proteins. On a basic level, both the GDI and Tiam1 form extensive contacts with the switch regions of the Rho protein (Fig. 4), and the binding of GDI will clearly be competitive with that of exchange factors. More fundamentally, these proteins engage a similar set of residues to exert opposing biochemical effects. All GTP-binding proteins require a Mg2+ ion for high affinity nucleotide binding. This Mg2+ ion offsets the negative charge of the phosphate groups to stabilize the bound nucleotide. Rho proteins exhibit unique coordination of this critical Mg2+ ion in the GDP-bound state,35 with the main-chain carbonyl of Thr35 directly coordinating the Mg2+ ion, replacing a water molecule found in the corresponding position of Ras. Importantly, the unique coordination of the Mg2+ ion by the main chain carbonyl of Thr35 provides access to the bound Mg2+ ion, allowing GDIs and GEFs to control nucleotide binding by influencing Mg2+ ion coordination. Specifically, the GDI interacts directly with the Thr35 side chain to stabilize the interaction of this residue with the Mg2+ ion and lock the nucleotide in the binding pocket, contacts mediated by a conserved aspartic acid and serine residues in the regulatory arm of the GDI (T47 and S47 in RhoGDI). As shown in (Fig. 4), Tiam1 forms main chain hydrogen bonding contacts on either side of this threonine residue, ratcheting switch I laterally along the nucleotide binding cleft, to pull the main chain carbonyl of Thr35 away from its position in the Cdc42/GDI complex. Disruption of the favorable interactions between Thr35 and the Mg2+ ion contributes to release of the bound nucleotide. In addition, Tiam1 disrupts the structure of the switch II domain of Rac such that the side chain of Ala59 protrudes into the Mg2+ ion binding site to assist in its ejection from the binding-pocket. Again, the GDI engages a similar set of residues in the switch II region to achieve the opposite effect, specifically fixing a conformation of switch II consistent with Mg2+ ion binding and stabilization of the associated GDP.

Figure 4. The involvement of threonine 35 in the regulation of nucleotide binding.

Figure 4

The involvement of threonine 35 in the regulation of nucleotide binding. Threonine 35 provides an important site for the regulation of nucleotide binding through the action of GEFs and GDIs. The Cdc42/GDI complex and the Rac/Tiam1 complex are shown oriented (more...)

Mechanism of GIP Activity

The GDI is unique among effectors and regulatory proteins in its ability to associate with both nucleotide states of Rho-family proteins. In the case of Cdc42, the affinity of the GDI for the GTP- and GDP-bound forms of the GTPase is identical.30 This ability to bind both conformations of the GTPase with equal affinity relies in part on the extensive contacts between the regulatory arm of the GDI and switch II. Relative to other members of the Ras superfamily, the nucleotide-dependent conformational changes in switch II are relatively subtle, thus allowing the GDI to bind to Cdc42 and presumably other Rho proteins in both nucleotide states.22

In the GTP-bound complex, the GDI is able to inhibit GTP hydrolysis. All of the GTPase/GDI structures solved to date involve a GDP-bound Rho protein, and while definitive proof of the mechanism of the GDI's GIP activity awaits a GTP-bound complex, some clues to the ability of the GDI to block GTP hydrolysis can be gleaned from comparison with GTPase/GAP complexes. Structures of Rho proteins in complex with their specific GAP proteins have been solved in both the ground state (GTP-bound)36 and transition state (aluminum fluoride-bound) conformations.37,38 These data demonstrate that GAP-catalyzed GTP hydrolysis proceeds by a two-pronged attack. First, the GAP introduces a catalytic arginine, the so-called "arginine-finger", into the active site. Second, the interaction of the GAP protein stabilizes a catalytically active conformation of the conserved glutamine residue in switch II (Gln61 in Cdc42). This glutamine residue is universally conserved in all GTP binding proteins and is absolutely required for GTP hydrolysis. In the Cdc42/GDI complex, GDI-binding rotates the side chain of the catalytic glutamine residue out of the nucleotide-binding pocket into an orientation that can no longer stabilize the transition state for GTP-hydrolysis. Thus, the binding of the GDI has essentially the same effect as mutating this critical catalytic residue. By subtly perturbing the structure of switch II, the GDI removes the catalytic glutamine from the active site into a position where it can no longer stabilize the transition state for GTP hydrolysis. Without this glutamine residue, hydrolysis can no longer proceed, allowing the GDI to maintain Rho-family proteins in a GTP-bound conformation. These structural rearrangements block intrinsic GTP-hydrolysis in the Cdc42/GDI complex. In addition, the GDI will clearly be competitive with GAP binding due to the extensive interface both proteins form with the switch regions of the Rho proteins, particularly switch II.

Structural Model for Membrane Release

The structure of the Cdc42/GDI complex represents the only structural data available for a fully processed, post-translationally modified GTP-binding protein, providing insight into the ability of these proteins to associate with biological membranes and the regulation of this process by the GDI. Upon complex formation, the geranylgeranyl-binding pocket in the immunoglobulin-like domain of the GDI expands to conform to the shape of the bound geranylgeranyl group. This complimentarity in shape is likely to provide specificity for binding to the geranylgeranyl group over other hydrophobic ligands. In addition, interactions between the polybasic tail of Cdc42 and an acidic patch on the surface of the GDI further ensure the specificity of the interaction. Kinetic data on the release of Cdc42 from cell membranes by the GDI suggests that this interaction proceeds by a two-step mechanism. On the basis of this biochemical work and the structural data, we propose the model for membrane release shown in (Fig. 5). In the absence of GDI binding, the geranylgeranyl group is inserted into the bilayer and the polybasic tail of Cdc42 interacts with the acidic head groups of the phospholipids. The interaction of the GDI with Cdc42 at the membrane surface corresponds to the initial rapid phase in the kinetic mechanism. Subsequently, a slower isomerization event occurs involving transfer of the geranylgeranyl group out of the bilayer and into the hydrophobic binding pocket of the GDI. This isomerization may be driven in part by competition between an acidic patch on the GDI and the acidic phospolipid headgroups for binding to the polybasic tail of Cdc42.

Figure 5. Structural model for membrane release.

Figure 5

Structural model for membrane release. A) A model, which accounts for the available structural and kinetic data,12 for GDI-mediated release of Rho family GTPases from cellular membranes is shown. The GTPase is localized to membranes by the insertion of (more...)

The importance of the GDI in regulating the cellular localization of Rho proteins makes the identification of small molecule inhibitors of the Cdc42/GDI interaction an attractive area of research. The geranylgeranyl-binding pocket is an obvious starting point for identifying such inhibitors and some progress has been made in isolating small molecules that bind in this hydrophobic pocket. Most of these analogs are geranylgeranyl cysteine analogs, which interact with relatively low (micromolar) affinity.39 The Cdc42/GDI complex has an affinity of 20 nM and, as such, the currently available small molecules will not act as efficient inhibitors of the Cdc42/GDI interaction. A recent finding highlighted the potential importance of carboxy-methylation demonstrating that carboxymethylated geranylgeranyl cysteine analogs bind to the GDI with much greater affinity than a corresponding unmethylated analog.39

The importance of methylation can be rationalized from the available structural data, which shows that the opening of the geranylgeranyl-binding pocket on the GDI is adjacent to a large acidic patch involved in interactions of the GDI with the polybasic tail of Cdc42. As described above, these interactions are thought to provide some of the driving force for extraction of Cdc42 from the membrane by competing for interactions of the polybasic tail with acidic phospholipid head groups. In an unmethylated situation, the acidic carboxy terminus would be directly adjacent to the acidic patch on the surface of the GDI, generating unfavorable electrostatic interactions between these two negatively charged regions. Methylation of the carboxyl group protects against such unfavorable electrostatic contacts and leads to a higher affinity interaction. Methylation presumably protects against similarly unfavorable interactions with the acidic head groups of phospholipids when the Rho proteins are inserted into the bilayer. The structural features of the geranylgeranyl-binding pocket seen in the Cdc42/GDI complex could provide an important starting point for designing higher affinity geranylgeranyl analogues for use as GDI inhibitors.

Future Directions

Regulation of the GTPase/GDI Complex

In the resting cell, a large fraction of Rho proteins are found in a cytosolic pool associated with the GDI.40,41 In this role, RhoGDI is primarily considered a negative regulator of signalling through Rho GTPases, and over-expression or microinjection of the GDI can be shown to block many signalling events mediated by Rho-family proteins.42-46 In order for Rho proteins to respond to activating signals, they must first be displaced from the GDI and inserted into cellular membranes. This event has been observed most dramatically in the case of RhoA activation in neuronal cells where stimulation with LPA causes its redistribution from the cytosol to the plasma membrane.47 The actin based morphology changes induced by RhoA activation in these cells depends on its membrane localization and can be blocked by GDI over-expression, emphasizing the importance of regulating the RhoA/GDI interaction in this process. Translocation to specific membrane-bound locations can also be demonstrated upon activation of Rho GTPases in other systems.48-51 While it is possible that simple mass action by Dbl-family GEFs is sufficient to displace the GDI and drive the association of Rho proteins with cellular membranes, the high affinity and 1:1 stoichiometry of the complex argues for the existence of a specific GDI displacement factor (GDF). A GDF would be capable of displacing the GDI and provides an appealing means for regulating the GTPase/GDI interaction. A GDF activity has recently been described for the regulation of the Rab/RabGDI interaction,52 and the identification of similar activities acting on Rho-family proteins is an important area of study.

The Ezrin-Radixin-Moeisin (ERM) family of proteins has been proposed to act as a GDF for the Rho proteins, regulating the Rho/GDI complex through direct interactions with the GDI.53 Binding of the amino-terminal domain of ERM proteins to RhoGDI causes the release of the GTPase from the complex allowing for its activation by GEFs (reviewed in ref. 54). The ERM proteins are actin-binding proteins involved in connecting the cytoskeleton with the plasma membrane and may play a role in the effects of Rho proteins on the actin cytoskeleton.55 In vivo, it appears that the ERM proteins are involved in Rho-mediated actin rearrangements, but many of these effects appear to be downstream of Rho activation. While the ERMs are candidates for GDI displacement factors involved in Rho signalling to the cytoskeleton, activated Rho proteins localize to a variety of cellular membranes and participate in signalling processes that do not involve ERM proteins, suggesting that there may be other GDFs acting in these pathways.

The Vav proto-oncogene was recently identified as specific GDI-binding partner, and may act as a GDF in certain Rac-dependent signalling pathways. Originally identified as a Dbl-family GEF specific for Rac, Vav was recently shown to interact directly with both RhoGDI and GDI2 through a calponin homology domain at its amino-terminus, a portion of the protein that is lost upon oncogenic activation.56 While the ability of Vav to regulate the Rac/GDI interaction has not been directly demonstrated, the coupling of a DH domain responsible for GEF activity with a GDI-binding domain raises the potential for coordinated down-regulation of the GDI binding and activation of nucleotide exchange. Phosphorylation of the RhoGDI downstream of phorbol ester stimulation of PKCα has also been shown to correlate with Rho activation, providing yet another potential mechanism for control of the GTPase/GDI interaction.57 Finally, the involvement of specific lipid products in controlling GDI release has been proposed.58 While ERM proteins remain the only proteins shown biochemically to dissociate Rho GTPases from a complex with the GDI, there is emerging evidence demonstrating that other signalling events impact directly on the GDI to modulate its interaction with Rho-family GTPases.

Positive Signalling Roles for the GDI

The cell maintains a careful balance in GDI expression, ensuring that there are nearly equal concentrations of the GDI and Rho proteins in the cell,15 and over-expression of the GDI may mask the more subtle regulatory features of its interaction with Rho GTPases. Recent work has pointed to a more complex role for GTPase/GDI complexes in directly mediating signalling events. Specifically, the Cdc42/GDI complex is able to block MAP-kinase activation downstream of Ras-GRF, suggesting a model in which Cdc42 and RhoGDI act to control signalling through the Ras/MAPK pathway.59 Disruption of the Cdc42/GDI complex and activation of Cdc42 releases the inhibition of Ras-GRF and is a prerequisite for Ras activation. In another study, the GDP-bound Rac/GDI complex can associate in the cytosol with a complex of the type I PIP-5 kinase and diacylglycerol kinase forming a preassembled signalling complex that translocates to the membrane upon GDI release,44 while the Rac/GDI complex is also able to effectively stimulate the NADPH-oxidase.25 Additional evidence for a positive signalling role for the GDI comes from the isolation of a Rac/GDI complex as a factor required for maintaining stimulated secretion in permeabilized mast cells.60 Interestingly, the GDI alone inhibited secretion, emphasizing the fact that over-expression of the GDI may mask its positive effects.

Conclusion

While structural studies on the GDI provide a mechanistic understanding of the regulation of the membrane association of Rho GTPases by the GDI, the signalling pathways that modulate this interaction are only now becoming clear. As described above, the importance of membrane localization in signalling by Rho GTPases, and the ability of GDIs to regulate this activity, point to an important role for the GDI in signalling through Rho family proteins. Further, the involvement of the GTPase/GDI complex in "positive" signals suggests that this complex represents an additional signalling state of the GTPase, in addition to the membrane associated GTP-bound form. The ability of the GDI to interact with both nucleotide states of the Rho GTPases suggests a mechanism in which the GDI may not only be involved in delivering the GDP-bound form of Rho proteins to the site of activation, but also in delivering the GTP-bound form to membrane compartments distant from the site of activation (Fig. 6). Interestingly, the membrane localization of certain Rho-family proteins in response to activating signals appears to be distinct from the "default" membrane localization seen in over-expression studies,15 suggesting that shuttling by the GDI may be important to accessing specific membrane bound locations in response to extracellular stimuli. The structural studies described here provide a mechanistic understanding of the biochemical activities of the GDI. Even more valuable will be the tools provided by the structure, particularly the ability to specifically disrupt formation of the GTPase/GDI complex and assess the role of the GDI in the varied signalling events mediated by Rho-family GTPases.

Figure 6. Model for the GDI as a shuttle in Cdc42 signalling.

Figure 6

Model for the GDI as a shuttle in Cdc42 signalling. The GDI protein, by virtue of its GIP activity, may act to deliver the activated form of Cdc42 to membrane locations distant from the site of activation. Similarly, the GDI can then act to recycle Cdc42 (more...)

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