Persistence without turnover: the RhoG G12E mutant highlights the role of nucleotide cycling in RhoG signaling

Small GTPases of the Ras superfamily act as molecular switches cycling between GDP-bound inactive and GTP-bound active states. Their signaling output depends not only on GTP loading but on continuous nucleotide cycling controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). While Glycine 12 substitutions in Ras typically confer constitutive activation, the effects of equivalent mutations in Rho-family GTPases remain poorly defined. Here, we characterize a ClinVar-reported RhoGG12E variant. Quantitative kinetic studies show that RhoGG12E has strongly impaired intrinsic and GAP-stimulated GTP hydrolysis but it can still be activated by GEFs. Effector binding is preserved in vitro and RhoGG12E accumulates in a GTP-bound state in cells. Yet, RhoGG12E induces enhanced spreading, increased focal adhesions, and reduced collective migration-phenotypes consistent with previously reported consequences of reduced RhoG signaling rather than hyperactivation. These findings indicate that accumulation of the GTP-bound form of small GTPases does not necessarily translate into productive output, supporting a model in which impaired nucleotide cycling compromises RhoG-dependent cellular behaviors.