Pandit S D, O'Hare T, Donis-Keller H, Pike L J
Department of Surgery, Division of Human Molecular Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Jan 24;272(4):2199-206. doi: 10.1074/jbc.272.4.2199.
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.
RET（转染时重组）基因编码一种受体酪氨酸激酶同源物，参与肠道神经支配和肾脏发育。构建了一种嵌合型表皮生长因子受体（EGFR）/RET受体，其包含与RET细胞内结构域融合的EGF受体的细胞外和跨膜结构域。该构建体在NIH 3T3细胞中表达，对该受体的功能特性进行了表征，并与野生型EGF受体的功能特性进行了比较。EGF受体对125I-EGF表现出高亲和力和低亲和力结合位点，而EGFR/RET嵌合体仅表现出对125I-EGF的低亲和力结合。该嵌合体能够比野生型EGF受体更快地内化EGF，并以EGF受体两倍的速率循环回到细胞表面。脉冲追踪实验表明，EGF刺激野生型EGF受体的降解，但对EGFR/RET受体的降解速率没有影响。循环增加和降解减少的组合导致EGFR/RET嵌合体的下调相对低效。用佛波醇12-肉豆蔻酸酯13-乙酸酯处理表达野生型EGF受体的细胞，导致125I-EGF结合减少和EGF刺激的酪氨酸磷酸化丧失。然而，佛波醇12-肉豆蔻酸酯13-乙酸酯处理对表达EGFR/RET嵌合体的细胞中的EGF结合和EGF刺激的酪氨酸激酶活性仅具有有限的影响。这些发现表明，Ret酪氨酸激酶不受许多用于通过生长因子受体终止信号传导的常见机制的调节。Ret酪氨酸激酶的这种持续激活可能与Ret在正常表达这种生长因子受体的细胞中的生理功能相关。
