Epidermal growth factor receptor internalization rate is regulated by negative charges near the SH2 binding site tyr992

This study examines the effects of mutations at and in the vicinity of tyro sine 992 of the epidermal growth factor receptor (EGFr) on epidermal growth factor- (EGF-) stimulated internalization of the receptor. Two regions of the EGFr adjacent to this domain have been defined previously as internaliz ation domains. The present work shows that the; mutation of negatively char ged amino acid residues near Tyr992 to their uncharged analogues increases the rate of EGF receptor internalization. In addition, the conversion of Ty r992, which is an EGFr ligand-induced autophosphorylation site, to phenylal anine also increases the rate of receptor internalization. However, the mut ation of Tyr992 to a glutamate residue does not alter the receptor internal ization rate. In addition, the truncation of the EGFr at glutamate 996 redu ces the internalization rate by half. This result confirms previous reports that residues immediately C-terminal to Glu996 are necessary to allow the normal rate of ligand-induced receptor endocytosis. The data suggest that n egative charge in the vicinity of Tyr992, and potentially the phosphorylati on of the EGFr at Tyr992, reduces the rate of ligand-induced receptor endoc ytosis. This reduction in internalization rate increases the lifetime of th e activated EGFr in the plasma membrane by about 70%, thus suggesting that phosphorylation of Tyr992 acts to increase the signaling capacity of the EG F receptor even as it directly acts as an SH2 binding site.