Kinetic and cell-based analyses of GTPase regulators

Regulatory GTPases are often portrayed as binary molecular switches that control a wide range of cellular processes, including, but not limited to, the generation of second messengers (e.g., cAMP and inositol phosphates), intracellular traffic, cytoskeleton organization and cell proliferation. GEF stimulators and GAP inhibitors regulate the nucleotide-bound state of these proteins. Because of the relevance of GTPases and their regulators to human diseases, they comprise a major therapeutic target. Currently, most of the information about GTPase regulators comes from structure analyses. Such structural information is limited to certain conditions and does not necessarily reflect specificity or physiological activity. To address questions about specificity and mechanisms of action, kinetic and cell-based analyses of GTPase regulators is necessary. Here, we compare these two approaches in the context of regulators of Arf and heterotrimeric G-proteins.