Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.     

Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation

To assess the functional significance of phosphorylation of the epidermal growth factor (EGF) receptor at Thr654, we compared the effects of 12-O-tetradecanoyl-13-acetate (TPA) on ligand-induced internalization and down-regulation between wild-type and mutant receptors that contain an alanine substitution at position 654. Activation of protein kinase C with TPA blocked EGF-induced internalization and down-regulation of Thr654 receptors and inhibited in vivo tyrosine kinase activity by 80%. TPA did not inhibit transferrin receptor internalization or constitutive EGF receptor internalization, suggesting that protein kinase C activation inhibits only the ligand-induced process. Inhibition by TPA of induced internalization, down-regulation, and kinase activity required threonine at position 654 since full-length Ala654 EGF receptors were significantly resistant to TPA inhibition of these ligand-induced activities. However, C'-terminal truncation further enhanced this resistance to TPA inhibition. The EGF-dependent internalization of kinase-inactive receptors truncated at residue 1022 was also impaired by TPA in Thr654 receptors, but not in Ala654 receptors, indicating that phosphorylation at Thr654 also interferes with tyrosine kinase-independent receptor activities. We conclude that the dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.     

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast     

EGF对EGFR基因和c-fos、c-myc原癌基因DNA结合蛋白质的影响

基因转录调控作用是EGF的细胞核内作用之一,而序列特异的DNA结合蛋白质与基因转录的调控密切相关。本文以C_3H小鼠胚胎正常成纤维细胞C_3H_(10)T_(1/2)C18(简称NC_3H_(10))株及其氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC_3H_(10))为对象,我们研究了EGF作用下,核内蛋白质与EGFR基因和c-fos、c-myc二种原癌基因的结合状态。研究发现,EGF的持续作用可使EGFR基因的特异结合蛋白质P120、c-fos基因的特异结合蛋白质P80、以及c-myc基因的特异结合蛋白质P125等明显增加。这些结果提示基因的特异结合蛋白质可能与EGF对基因转录的调控密切相关。上述结果尚未见到国内外同类报道。     

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.