Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts.
Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.
Sturani
E
E
Department of General Physiology and Biochemistry, University of Milan, Italy.
Zippel
R
R
Morello
L
L
Brambilla
R
R
Comoglio
P M
PM
Alberghina
L
L
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D007295:Inositol Phosphates; D014443:Tyrosine; D066246:ErbB Receptors; D002118:Calcium