Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand-induced internalization of the epidermal growth factor receptor.
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.
Heisermann
G J
GJ
Department of Medicine, University of California, San Diego, La Jolla 92093.
Wiley
H S
HS
Walsh
B J
BJ
Ingraham
H A
HA
Fiol
C J
CJ
Gill
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047388:Casein Kinases; D002460:Cell Line; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000138:chemical synthesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D015183:Restriction Mapping; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D008024:Ligands; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D013912:Threonine; D012694:Serine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors; D047388:Casein Kinases