High Throughput strategies Aimed at Closing the GAP in Our Knowledge of Rho GTPase Signaling

Abstract

Since their discovery, Rho GTPases have emerged as key regulators of cytoskeletal dynamics. In humans, there are 20 Rho GTPases and more than 150 regulators that belong to the RhoGEF, RhoGAP, and RhoGDI families. Throughout development, Rho GTPases choregraph a plethora of cellular processes essential for cellular migration, cell-cell junctions, and cell polarity assembly. Rho GTPases are also significant mediators of cancer cell invasion. Nevertheless, to date only a few molecules from these intricate signaling networks have been studied in depth, which has prevented appreciation for the full scope of Rho GTPases' biological functions. Given the large complexity involved, system level studies are required to fully grasp the extent of their biological roles and regulation. Recently, several groups have tackled this challenge by using proteomic approaches to map the full repertoire of Rho GTPases and Rho regulators protein interactions. These studies have provided in-depth understanding of Rho regulators specificity and have contributed to expand Rho GTPases' effector portfolio. Additionally, new roles for understudied family members were unraveled using high throughput screening strategies using cell culture models and mouse embryos. In this review, we highlight theses latest large-scale efforts, and we discuss the emerging opportunities that may lead to the next wave of discoveries.

Keywords: Rho GTPase 1; RhoGAP 3; RhoGDI 4; RhoGEF 2; high throughput screening 5; proximity labelling 6.

Figure 1

Rho GTPases classification in mammals. Rho GTPases are divided into eight subfamilies, namely the CDC42, RAC, RHO, RHOF, RHOBTB, RHOH, RHOU/RHOV, and RND subfamilies. These are further divided between classical and atypical Rho GTPases. Classical Rho GTPases cycle between an inactive GDP and an activate GTP-bound state while atypical Rho GTPases are mainly regulated through other mechanisms.

Figure 2

The classical Rho GTPase cycle and its spatiotemporal regulation. RhoGEFs activate Rho GTPases by destabilizing Rho GTPase nucleotide and Mg²⁺ interaction and by promoting GTP loading, which is in excess in cells when compared to GDP. RhoGEFs also act as protein scaffolds by bringing appropriate effectors in close proximity to active Rho GTPases. Once active, Rho GTPases bind with their effectors and trigger biological effects in cells such as cell migration, cell adhesion, and cell polarity. RhoGAPs inactivate Rho GTPases by enhancing their weak intrinsic GTPase activity, which triggers the hydrolysis of GTP in GDP. RhoGDIs bind to Rho GTPases and promote the extraction of the Rho GTPases C-terminal lipid extension from the membrane which becomes ultimately hidden into the RhoGDIs hydrophobic pocket. RhoGDIs are known to sequester Rho GTPases in the cytosol and to prevent their interaction with other regulators. Rho GTPases are also heavily regulated through post-translational modifications, gene expression and their degradation.

Figure 3

Mapping of Rho GTPases regulators specificities. (a) RhoGEFs exhibit specificity towards few Rho GTPases. (b) Examples of RhoGEFs and their specificity towards Rho GTPases as mapped by proximity labeling coupled with mass spectrometry. (c) RhoGAPs are promiscuous and display broad activity towards Rho GTPases. (d) Examples of Rho GAPs and their specificity towards Rho GTPases as mapped by proximity labeling coupled with mass spectrometry.

Figure 4

The spatial distribution of Rho GTPases by their regulators the RhoGEFs and RhoGAPs determines Rho GTPase activity. Schematic representation showing that the spatial distribution of RhoGEFs and RhoGAPs helps to create Rho GTPases active and inactive zones in cells The Golgi and mitochondria only host RhoGAPs. This could allow the inactivation of Rho GTPases that would have diffused away from their desired site of action.