An atypical link between Wrch1/RhoU and multiple upstream signals is mediated through only one of its N-terminal SH3-domain binding motifs

Wnt-regulated Cdc42 homolog-1 (Wrch1) is an atypical member of the Rho GTPase family. A major difference is the N-terminal 46 amino acid extension containing three putative SH3 domain-binding motifs, whose specificities were not determined so far. In this study we analyzed Wrch1 interaction with SH3 domains of different adaptor proteins using sedimentation and isothermal titration calorimetry and found out Grb2, Nck1, Src, Crk and p120RasGAP as binding partners for Wrch1 N-terminus. Measurements with separate peptides related to the three putative SH3 domain binding motifs of Wrch1 showed that only the central PxxP motif is responsible for the adaptor protein binding and determined the minimal consensus sequence of Wrch1 central binding motif. Taken together, our data suggest unique properties of Wrch1 in comparison to conventional GTPases regarding its interaction with SH3 domain containing adaptor proteins and suggest multiple upstream signals converging on Wrch1 directly through its SH3 domain binding properties.