title: SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins.
abstract: Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.
authors.affiliation: Department of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, D-40225 Dusseldorf, Germany.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Risse
authors.forename: Belen
authors.initials: B
authors.lastname: Vaz
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Burton
authors.forename: Pontus
authors.initials: P
authors.lastname: Aspenström
authors.forename: Roland P
authors.initials: RP
authors.lastname: Piekorz
authors.forename: Luc
authors.initials: L
authors.lastname: Brunsveld
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Ahmadian
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C078332:Nck protein; D015513:Oncogene Proteins; C432668:RHOU protein, human; D020741:rho GTP-Binding Proteins