Release of the neuregulin functional polypeptide requires its cytoplasmic tail

Based on both in vivo and in vitro studies, we have shown previously that t he intracellular domain of a membrane-bound isoform of the growth factor, n euregulin, regulates proteolytic release of its extracellular domain ErbB r eceptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfe ction in COS-7 cells. These studies revealed that regulation of extracellul ar domain cleavage by the intracellular domain is sequence-specific and inv olves three distinct 30-60-residue segments. The presence of any two of the se three segments is both necessary and sufficient for proteolytic processi ng, and resistance to proteolysis is not due to an alteration in cellular l ocalization or transport. Evidence was also obtained that regulation of ext racellular domain processing involves initial intracellular domain dimeriza tion. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Fu rthermore, resistance to proteolytic processing of a construct lacking a la rge portion of the intracellular domain was rescued with a chimera, in whic h the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain i nteractions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms.