Fig. 5. Rho1 interacts directly with p120^ctn and -catenin. (A) GST pulldown experiments assessing binding among Rho1, the DE-cadherin intracellular domain, p120^ctn, ß-catenin and -catenin. ³⁵S-labeled in vitro translated (IVT) -catenin (third panel from top) binds GST-Rho1 independently of the phosphorylation state of its associated nucleotide, IVT-p120^ctn binds preferentially to GST-Rho1^GDP (top panel) and IVT-ß-catenin does not interact with either form of GST-Rho1 (second panel from top). Rok and a putative RhoGDI are included as binding controls. 5% input is shown. (B) GST pulldown experiment utilizing purified bacterially-expressed His-p120^ctn. His-p120^ctn also binds preferentially to GST-Rho1^GDP. (C) Immunoprecipitations showing in vivo interaction between Rho1 and -catenin, and Rho1 and p120^ctn. ß-catenin immunoprecipitations were performed as a positive control. 5% input is shown. (D) Diagram of the protein fragments, substitutions and point mutants used to map interaction domains on Rho1. (E) Computer model of GDP-bound RhoA crystal structure. Residues required for -catenin binding are shownin yellow and those required for p120^ctn binding are shown in red. For reference the effector domain is highlighted in green. (F) GST pulldown experiments demonstrating the regions of Rho1 required for binding of -catenin (top panel) and p120^ctn (second panel from top). -catenin binds to region A and its binding is disrupted by replacing aa 27-29 (KDQ) with alanines, whereas p120^ctn binds to region C and its binding is disrupted by replacing aa 51-54 (KQVE) with alanines. Rok and RhoGDI bound preferentially to constitutively active (V14A) and dominant negative (N17A) forms of Rho1, respectively, but equally well to all other forms of Rho1 tested. V14A protein was exchanged with GTP and N17A with GDP in all experiments. All other forms of Rho1 in the -catenin, p120^ctn and RhoGDI binding experiments were exchanged with GDP, while the Rho1 proteins used to test Rok binding were exchanged with GTP.