TI - Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand -induced internalization of the epidermal growth factor receptor . AB - The epidermal growth factor ( EGF ) receptor contains multiple sites of phosphorylation on serine , threonine and tyrosine residues . Because the biological responsiveness of the EGF receptor is regulated by phosphorylation at several of these sites , we studied the functional consequences of removal of the Thr669 and Ser671 phosphorylation sites using site -directed mutagenesis . The mutant EGF receptor expressed in mouse B82 cells displayed normal EGF binding and in vivo autophosphorylation and was fully active in biological signal transduction as measured by EGF -stimulated gene transcription . However , the EGF -dependent phosphorylation of an 85-kDa cellular substrate by the mutant receptor was impaired relative to the wild type receptor , indicating that the mutated region may specifically interact with this substrate . Endocytosis of the mutant receptor was also impaired as measured by both receptor down-regulation and ligand internalization studies . This was due to impaired uptake of the mutant receptor by the saturable , high affinity endocytic system . Several aspects of mutant receptor function were regulated normally by TPA , indicating a lack of interaction between the mutated phosphorylation sites and the nearby protein kinase C phosphorylation site Thr654 . These results suggest that phosphorylation of the EGF receptor at Thr669 and Ser671 mediates interaction of the receptor with a specific tyrosine kinase substrate and is required for efficient ligand -induced receptor internalization .