Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines.
The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.
Helin
K
K
Institute of Microbiology, University of Copenhagen, Denmark.
Beguinot
L
L
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases