IQGAP1 interaction with RHO family proteins revisited: Kinetic andequilibrium evidence for multiple distinct binding sites

IQ motif-containing GTPase activatingprotein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity and transmigration. The RHO family proteins CDC42 and RAC1, have been shown to mainly interact with the GAP- related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal (CT) domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underly a multiple-step binding mechanism: (i) a high-affinity, GTP- dependent binding of RGCT to the switch regions of CDC42 or RAC1, and (ii) a very low-affinity binding of GRD and CT adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integrat on ofIQGAP1-mediated cellular responses.