BINDING-SPECIFICITY AND IN-VIVO TARGETS OF THE EH DOMAIN, A NOVEL PROTEIN-PROTEIN INTERACTION MODULE

EH is a recently identified protein-protein interaction domain found i n the signal transducers Eps15 and Eps15R and several other proteins o f yeast nematode. We show that EH domains from Eps15 and Eps15R bind i n vitro to peptides containing an asparagine-proline-phenylalanine (NP F) motif. Direct screening of expression libraries with EH domains yie lded a number of putative EH interactors, all of which possessed NPF m otifs that were shown to be responsible for the interaction. Among the se interactors were the human homolog of NUMB, a developmentally regua ted gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a fam ily of EH-containing proteins in mammals. Eased on the characteristics of EH-containing and ED-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.