We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca(2+)-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.