## 1 Introduction  
RHO GTPases act, with some exceptions [1], as molecular switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Their functions at the plasma membrane are usually controlled by three groups of regulatory proteins: guanine nucleotide dissociation inhibitors (GDIs), guanine nucleotide exchange factors (GEFs) and GTPases activating proteins (GAPs) [2]. The formation of the active GTP-bound state of -RHO GTPases, such as CDC42, is accompanied by a conformational change in two regions, known as switch I and II (encompassing amino acids or aa 29-42 and 62-68, respectively); these regions provide a platform for a GTP-dependent, high-affinity association of structurally and functionally diverse effector proteins, e.g., ACK, PAK1, WASP, ROCKI, DIA and IQGAP1, through their so-called GTPase-binding domains (GBDs) [3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13] (reviewed in [14]). GTPase-effector signaling activates further a wide variety of pathways in all eukaryotic cells [2]. A unique feature distinguishing the RHO family from other small GTPase families is the presence of a 12 amino-acid insertion (aa 124-135 in CDC42) that protrudes from the G domain structure by forming a short helix, the so-called insert helix (IH) [15]. This IH is highly charged and variable among the members of the RHO family [15]. The IH has been very recently shown to have larger conformational flexibility in the GDP-bound CDC42 than in the GTP-bound CDC42 [16]. IH is a binding site for RHOGDI1, p50 GAP, DIA, FMNL2, PLD1 and IQGAP2 [10; 12; 17; 18; 19; 20; 21], and appears rather essential for downstream activation of RHO GTPases [22; 23; 21]. IQGAP1 is ubiquitously expressed and shares a similar domain structure with its human paralogs IQGAP2 and IQGAP3 (Figure 1A), including an N-terminal calponin homology domain (CHD), a coiled-coil repeat region (CC), a tryptophan-containing proline-rich motif-binding region (WW), four isoleucine/glutamine-containing motifs (IQ), a RASGAP-related domain (GRD), a RASGAP C-terminal domain (RGCT) and a very C-terminal domain (CT). IQGAPs interact with a large number of proteins and modulate the spatiotemporal distributions of distinct signal-transducing protein complexes [24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34]. As multidomain scaffold proteins, they safeguard the magnitude, efficiency and specificity of signal transduction [35]. They have been localized at multiple subcellular sites orchestrating different signaling pathways and thus controlling a variety of cellular functions [36; 37; 38; 39; 40; 41; 42]. Notably, IQGAP1 has been implicated as a drug target due to its vital regulatory roles in cancer development [43; 44; 45; 46; 47; 48; 49] although the molecular mechanism of its functions is unclear. Figure 1: IQGAP GRD binding is nucleotide independent. (**A**) Domain organization of the IQGAP paralogs along with their GRDs and C-terminal fragments assessed in this study (see text for more details). (**B**) Fluorescence polarization analysis of IQGAP1 and IQGAP2 proteins with mGppNHp- and mGDP-bound CDC42. (**C**) Pull-down of endogenous IQGAP1 FL from HEK293 lysates with GppNHp- or GDP-bound GST-CDC42 and GST-RAC1, respectively. Densiometry evaluation of relative IQGAP1 binding to GST-CDC42 proteins (a. u., arbitrary unit) from a triplicate experiment is shown as bar charts. Earlier studies analyzed the crucial role of IQGAP RGC in high-affinity binding to the switch regions of the GTP-bound, active CDC42 and proposed it as an IQGAP 'effector domain' [5; 50; 51]. Accordingly, [PERSON] et al. reported that the CDC42 GppNHp can still bind to IQGAP-\(\Delta\)GRD (lacking aa 1122-1324) [52]. Moreover, [PERSON] et al. mapped the CDC42 and IQGAP binding regions and determined that switch I and surrounding regions (residues 29-55) together with the insert region (residues 122-134) are required for high affinity binding to IQGAP1 [53].