Functional characterization of an epidermal growth factor receptor/RET chimera.
The RET (recombined in transfection) gene encodes a receptor tyrosine kinase homolog involved in innervation of the gut and renal development. A chimeric epidermal growth factor receptor (EGFR)/RET receptor was constructed which contained the extracellular and transmembrane domains of the EGF receptor fused to the intracellular domain of RET. This construct was expressed in NIH 3T3 cells, and the functional properties of the receptor were characterized and compared with those of the wild type EGF receptor. Whereas the EGF receptor exhibited both high and low affinity binding sites for 125I-EGF, the EGFR/RET chimera exhibited only low affinity binding of 125I-EGF. The chimera was able to internalize EGF more rapidly than the wild type EGF receptor and recycled to the cell surface at twice the rate of the EGF receptor. Pulse-chase experiments indicated that EGF stimulated the degradation of the wild type EGF receptor but had no effect on the rate of degradation of the EGFR/RET receptor. The combination of increased recycling and decreased degradation resulted in the relatively inefficient down-regulation of the EGFR/RET chimera. Incubation of cells expressing the wild type EGF receptor with phorbol 12-myristate 13-acetate led to a reduction in 125I-EGF binding and a loss in EGF-stimulated tyrosine phosphorylation. However, phorbol 12-myristate 13-acetate treatment had only a limited effect on EGF binding and EGF-stimulated tyrosine kinase activity in cells expressing EGFR/RET chimeras. These findings suggest that the ret tyrosine kinase is not regulated by many of the common mechanisms used to terminate signaling via growth factor receptors. Such persistent activation of the Ret tyrosine kinase may be relevant to the physiological function of Ret in cells that normally express this growth factor receptor.
Pandit
S D
SD
Department of Surgery, Division of Human Molecular Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
O'Hare
T
T
Donis-Keller
H
H
Pike
L J
LJ
D016475:3T3 Cells; D000818:Animals; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008934:Mitogens / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D008934:Mitogens; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases