Abstract
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.
Highlights
The RET gene encodes a receptor tyrosine kinase homolog [1, 2]
Previous studies of epidermal growth factor receptor (EGFR)/RET chimeras have demonstrated that the chimeras respond to EGF and mediate the activation of a variety of downstream signaling pathways including the activation of phospholipase C, Ras, and MAP kinase [18, 19]
The presence of two classes of EGF binding sites was readily demonstrable in cells transfected with the wild type EGF receptor, cells expressing the EGFR/RET chimera consistently showed only a single class of low affinity binding sites for EGF
Summary
The RET gene encodes a receptor tyrosine kinase homolog [1, 2]. RET was first identified in DNA transfection studies in which the gene had been recombined in transfection to generate a fusion protein that induced the transformation of NIH 3T3 cells [3]. Subsequent studies have shown that activating mutations in the RET gene are responsible for several dominantly inherited human neoplasias including multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma (4 – 8). Van Weering et al [19] expressed a similar EGFR/RET chimera in SK-N-MC cells, a neuroectodermal cell line In this system, EGF stimulated the tyrosine kinase activity of the EGFR/RET chimera and led to the sustained activation of MAP kinase. Despite the fact that EGF failed to stimulate MAP kinase in 3T3 cells expressing the EGFR/RET chimera, this growth factor did induce both proliferation and transformation of these cells [18], suggesting that activation of MAP kinase may not be required for Ret to promote proliferation of 3T3 cells. The absence of such termination events suggests that prolonged activation of Ret and/or a maintenance of sensitivity to Ret ligand may be physiologically important for cells that normally express this growth factor receptor
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