Cytoskeleton

Cell

GTPase

Moesin

RHOA

Ezrin

Membrane ruffling

Effector

Biology

Guanine nucleotide exchange factor

Radixin

RAC1

Rac GTP-Binding Proteins

Signal transduction

Cell biology

Genetics

Nature Cell Biology

Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin–Radixin–Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family. Using a BioID approach, Bagci et al. systematically analyse the Rho-family GTPase interactome and reveal previously unappreciated interactions with RhoGEFs and RhoGAPs and effectors for Rho proteins.

Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms