The IQGAP1-Rac1 and IQGAP1-Cdc42 Interactions

Abstract

IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/ Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/ IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.The Rho family of small G proteins are well documented to regulate cell motility through their regulation of the actin cytoskeleton (1). These small G proteins act as molecular switches, being active in the GTP-bound form and inactive in the GDP-bound form. The interaction of the active, GTPbound, G protein with an effector protein triggers a downstream signaling cascade. Rho family proteins, acting via these effector proteins, have been shown to control the polymerization of actin in the peripheral cortex of the cell. Cdc42 controls filopodia formation; these are actin-rich fingers that establish the direction of motility. Rac1 controls lamellipodia formation; these actin-rich ruffles at the leading edge of the cell initiate motion, whereas Rho controls the establishment of stress fibers whose formation results in the contractile force that moves the body of the cell behind the leading edge (2).Effector proteins for two members of the Rho family, Cdc42 and Rac1, include the PAK 3 family of serine/threonine kinases reviewed in Ref. 3, the ACK tyrosine kin...

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A novel computer simulation method for simulating the multiscale transduction dynamics of signal proteins

“…This causes that the steric inhibition from the LOV2 core is relieved and the activation site of Rac1 becomes accessible to interactions with its effector domains, such as PAK1. [74][75][76] By contrast, we infer from Fig. 8(a) that in case of the dark state the switchII region is blocked by the Aβ-Bβ-loop, which causes that the Rac1 enzyme is inactive under dark-state conditions.…”

Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

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“…Presumably, increased affinity toward GDP-bound Cdc42 by full-length IQGAP1 (versus the isolated GRD) is afforded by sequences outside the GRD that also increase affinity toward active Cdc42. This was observed for the larger IQGAP1 fragment (residues 864 -1657), which binds active Cdc42 with ϳ50-fold higher affinity than the isolated GRD (45).…”

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