The focus of this application is to explore a novel pathway for the proteolytic processing of receptor tyrosine kinases. In particular, this involves the two step cleavage of the ErbB-4 receptor that binds the growth factor heregulin. Cleavage of ErbB-4 can be stimulated by heregulin or TPA and results in the relocalization of the ErbB-4 cytoplasmic domain, including its tyrosine kinase, from the plasma membrane to the nucleus. The application seeks to elucidate the biochemical parameters of the two cleavage events, as well as the biological significance of the processing pathway for ErbB-4 mediated cellular responses. The first aim will characterize the initial and essential secretase (metalloprotease)-dependent cleavage of the ErbB-4 ectodomain. This will include identification of the cleavage site, construction of receptor and protease mutants, evaluation of the contributions of ErbB-4 structure to protease recognition, and the relationship of cleavage and signal transduction to heregulin-induced translocation of ErbB-4 to membrane microdomains. The second aim will concentrate on the subsequent gamma-secretase (presenilin) mediated cleavage that releases the ErbB-4 cytoplasmic domain from the plasma membrane. This will include analysis of the gamma-secretase cleavage site, the role of PDZ domain proteins and presenilins, and nuclear translocation of the ErbB-4 cytoplasmic domain. The final aim is constructed to address the physiological significance of ErbB-4 cleavage and nuclear re-localization of the cytoplasmic domain. This will include cell biological experiments of cytoplasmic domain functions in the nucleus, such as tyrosine phosphorylation and the activation of gene expression. Selective inhibitors of proteolytic processing will be used to assess the role of this pathway in ErbB-4 mediated biological responses. Lastly, a mouse "knockin" approach is proposed to assess the physiological significance of this novel receptor processing pathway in the animal.