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 GEFs and GAPs. While Gly12 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 RhoG ^G12E variant. Quantitative kinetic studies show that RhoG ^G12E has strongly impaired intrinsic and GAP-stimulated GTP hydrolysis but it can still be activated by GEFs. Effector binding is preserved in vitro and RhoG ^G12E accumulates in a GTP-bound state in cells. Yet, RhoG ^G12E 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.