mGrb10 interacts with Nedd4

We have utilized the yeast two-hybrid system to identify proteins interacti ng with mouse Grb10, an adapter protein known to interact with both the ins ulin and the insulin-like growth factor-I receptors. We have isolated a mou se cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a beet (homologous to the EG-AP carboxyl-ter minus) domain and three WW domains. The interaction with Grb10 in the two-h ybrid system was confirmed using the full-length Nedd4, and it was abolishe d 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 Gr b10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, wher e endogenous Nedd4 co-immunoprecipitated constitutively with both the endog enous and an overexpressed Grb10. This interaction was Ca2+-independent. Gr b10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possi bility that this interaction may be used to target other proteins, like tyr osine kinase receptors, for ubiquitination.