GTP-dependent assembly of an IQGAP2/arp2/3-actin scaffold.

(A) The ability of GST-PBD to specifically bind IQGAP2 was determined by cDNA cotransfection studies in COS1 cells using IQGAP2 and constitutively active rac1V12 (lane 1) or cdc42V12 (lane 3) or the dominant-negative rac1N17 (lane 2) or cdc42N17 (lane 4) mutants, followed by bead-affinity assays and immunoblot analysis with anti-IQGAP2 or anti-GTPase antibodies. GST-conjugated agarose beads lacking the PBD domain failed to precipitate [GTP]rac1/IQGAP2 or [GTP]cdc42/IQGAP2 complex (not shown). (B) GFPs were resuspended to 2.5 × 10⁸/mL, and 200-μL aliquots were activated with individual agonists for various time points as outlined. Samples were then solubilized in RIPA (final concentration, 100 μg/sample), incubated with GST-PBD, and evaluated by SDS-PAGE and immunoblot analysis for the presence of IQGAP2, arp3a, arp2, or actin. (C) F-actin content determinations for individual agonists; F-actin content is inhibited in thrombin-stimulated platelets preincubated with 10 μM cytochalasin D (Cyt D). (D) Percentage (of total) of individual proteins identified within the thrombin-induced IQGAP2 scaffold was determined by densitometric comparison to 5 μg total platelet lysates, adjusted for nonspecific binding using parallel samples incubated with equivalent concentrations of GST control beads. Note that neither gelsolin nor WASp is identified within these complexes and that preincubation of platelets with 10 μM cytochalasin D abrogates thrombin-induced IQGAP2/actin association. Results are representative of one complete set of experiments completed on 2 separate occasions from 2 different healthy volunteers.