RhoGDI: multiple functions in the regulation of Rho family GTPase activities

Abstract

RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase-RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.

Figure 1. Cdc42–RhoGDIα interactions

(A) Overview of the Cdc42–RhoGDIα complex. Cdc42 is shown in brown and its isoprenyl moiety in white space-fill (prenyl). The nucleotide (GDP) and the Mg²⁺ ion, as well as the switch I and II regions (swI and swII respectively), of Cdc42 are also indicated. The N-terminal domain of RhoGDIα is in yellow with the helix–loop–helix motif indicated through helices α2 and α3. The C-terminal immunoglobulin-like motif is in blue. (B) Nucleotide binding occurs through co-ordination of the Mg²⁺ ion by the main chain carbonyl group of Thr³⁵_(Cdc42). RhoGDIα stabilizes the GDP-bound form and prevents nucleotide dissociation through hydrogen bonds (dashed green lines) occurring between the carboxy oxygen of Asp⁴⁵_(RhoGDI) and the hydroxy group of Thr³⁵_(Cdc42) and between the hydroxy group of Ser⁴⁷_(RhoGDI) and the main chain amide and carbonyl groups of Val³⁶_(Cdc42). (C) Overview of the hydrophobic pocket of RhoGDIα. Shown is the α–carbon atom trace. Conserved hydrophobic amino acids (red) that form favourable van der Waals contacts along the length of the geranylgeranyl group (dark blue) line the pocket. Also indicated is the C-terminal hypervariable region of Cdc42 (brown). (D) Arg⁶⁶_(Cdc42) constitutes an important residue for the interaction with RhoGDIα as it can interact with amino acids from both the N- and C-terminal domains of RhoGDIα. The hydrogen bonds between the side chain of Arg⁶⁶_(Cdc42) and the main chain carbonyl group of Ala³¹_(RhoGDI) and the side chain of Asp¹⁸⁵_(RhoGDI) are indicated. Also shown is the highly conserved interaction between Asp¹⁸⁴_(RhoGDI) and His¹⁰³_(Cdc42). Mutation of either of these residues on GTPases results in RhoGDI-binding deficiency [–60,62,66].

Figure 2. Sequence alignments of the three human RhoGDIs (A) and the three family-defining RhoGTPases (B)

*, identical residues; :, conservation of homologous residues;·, strong conservation of groups. (A) Elements of secondary structure are indicated above the sequences according to Hoffman et al. [30], except for helix α1, assigned according to Golovanov et al. [28]. ● indicates residues important for the protein–protein interface, as determined from the three crystal structures [–32]. ○ indicates residues lining the hydrophobic pocket as determined by Hoffman et al. [30]. The single amino acid difference between RhoGDIα and RhoGDIβ responsible for their difference in affinity towards Cdc42 [15] is boxed. (B) Residues of Rho GTPases contributing to the interactions with RhoGDIs as determined by the three crystal structures are found mostly in and proximal to the switch regions and the C-terminal hypervariable sequences and are boxed in grey. The insert region is also indicated. The serine residues on RhoA (Ser¹⁸⁸) and Cdc42 (Ser¹⁸⁵), shown in bold, are substrates for PKA and their phosphorylation contributes to enhanced association with RhoGDIα [36,37].

Figure 3. Models for the regulation of Rho family GTPases by RhoGDI molecules

(A) GDP-bound Rho is complexed to RhoGDI in the cytosol. A displacement factor or signal (e.g. ERM family members or a kinase such as PKCα or PAK) localizes the complex proximal to a membrane compartment (1) and releases the GTPase from RhoGDI (2). Exchange of GDP for GTP is catalysed by a GEF protein (3) and allows the GTPase to associate with effector proteins and propagate its signal (4). GTP hydrolysis facilitated by a GAP protein terminates the signal and allows membrane extraction by RhoGDI (5). (B) Once nucleotide exchange is performed (3), RhoGDI might extract the GTPase from the membrane in its GTP-bound form (6) to either terminate the signal prematurely (e.g. following phosphorylation by PKA) or to redirect the GTPase to a distinct membrane compartment within the cell (7). This might be achieved by prior association with an effector protein or a component of a larger protein complex (8,9) and through the assistance of specific localization signals inherent to the particular membrane domain (e.g. a specific membrane-lipid enrichment).