Determination of the Activity of Rho-Like GTPases in Cells

Rho-like GTPases, including RhoA, Rac1 and Cdc42, have been implicated in the control of a wide range of biological processes such as the organization of cytoskeletal structures, adhesion, motility, transcriptional activation, and cell-cycle progression (1 –3 ). The Rho-like GTPases comprise a subfamily of the Ras superfamily of small guanosine triphosphate (GTP)-binding proteins. Like all members of the Ras superfamily, the Rho-GTPases function as molecular switches, cycling between an inactive guanosine diphosphate (GDP)-bound state and an active GTP-bound state. Among all Ras superfamily members, specific guanine-nucleotide-exchange factors (GEFs) activate Rho-like GTPases by promoting the transition from the GDP-bound to the GTP-bound state, while GTPase-activating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP, inactivate Rho-like proteins. Rho-like GTPases are further regulated by guanine-nucleotide dissociation inhibitors (GDIs), which can inhibit both the exchange of GTP and the hydrolysis of bound GTP. Rho GDIs, as well as members of the ERM family (ezrin, radixin, and moesin), also appear to play a crucial role in the translocation of Rho-like GTPases between membranes and the cytoplasm-a process also associated with their activation. During recent years, numerous downstream effector proteins that bind Rho, Rac, and Cdc42 in their GTP-bound form have only been identified in an attempt to reveal the underlying molecular mechanisms by which the Rho-like GTPases mediate their various effects.