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 developmen t, A chimeric epidermal growth factor receptor (EGFR)/RET receptor was constructed which contained the extracellular and transmembrane domai ns of the EGF receptor fused to the intracellular domain of RET. This construct was expressed in NIH 3T3 cells, and the functional propertie s of the receptor were characterized and compared with those of the wi ld type EGF receptor, Whereas the EGF receptor exhibited both high and low affinity binding sites for I-125-EGF, the EGFR/RET chimera exhibi ted only low affinity binding of I-125-EGF, The chimera was able to in ternalize EGF more rapidly than the wild type EGF receptor and recycle d to the cell surface at twice the rate of the EGF receptor. pulse-cha se experiments indicated that EGF stimulated the degradation of the wi ld type EGF receptor but had no effect on the rate of degradation of t he EGFR/RET receptor. The combination of increased recycling and decre ased degradation resulted in the relatively inefficient down-regulatio n of the EGFR/RET chimera. Incubation of cells expressing the wild typ e EGF receptor with phorbol 12-myristate 13-acetate led to a reduction in I-125-EGF binding and a loss in EGF-stimulated tyrosine phosphoryl ation. However, phorbol 12-myristate 13-acetate treatment had only a l imited effect on EGF binding and EGF-stimulated tyrosine kinase activi ty in cells expressing EGFR/RET chimeras. These findings suggest that the ret tyrosine kinase is not regulated by many of the common mechani sms used to terminate signaling via growth factor receptors, Such pers istent activation of the Ret tyrosine kinase may be relevant to the ph ysiological function of Ret in cells that normally express this growth factor receptor.