Huang Yao, Chang Yongchang, Wang Xiangdong, Jiang Jing, Frank Stuart J
Department of Medicine, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012, USA.
Endocrinology. 2004 Jul;145(7):3297-306. doi: 10.1210/en.2003-1658. Epub 2004 Apr 7.
Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of (125)I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH's effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR's postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.
表皮生长因子受体（EGFR）是一种跨膜蛋白，其胞外结构域可结合表皮生长因子（EGF），并通过其胞质结构域内的内在酪氨酸激酶活性启动信号传导。EGFR在发育、细胞增殖和癌症中起着重要作用。生长激素（GH）是一种关键的促进生长和调节代谢的激素，它与生长激素受体结合，从而激活包括细胞外调节蛋白激酶（ERK）在内的各种信号通路。先前的研究表明生长激素受体和表皮生长因子受体信号系统之间存在相互作用。利用对生长激素和表皮生长因子有反应的3T3-F442A前脂肪细胞，我们先前观察到，生长激素除了引起表皮生长因子受体酪氨酸磷酸化外，还诱导了可被PTP101检测到的表皮生长因子受体磷酸化，PTP101是一种与ERK共有磷酸化位点反应的抗体。后一种磷酸化可通过用丝裂原活化蛋白激酶（MEK）1抑制剂预处理来阻止，这表明其依赖于ERK途径。此外，生长激素与表皮生长因子共同处理显著减缓了表皮生长因子诱导的表皮生长因子受体降解和下调，从而增强了表皮生长因子诱导的表皮生长因子受体信号传导。这些效应也依赖于MEK1，表明生长激素对表皮生长因子诱导的表皮生长因子受体胞吞后运输和信号传导具有ERK途径依赖性影响。我们现在探讨生长激素对3T3-F442A细胞中表皮生长因子细胞表面结合的影响。我们发现，生长激素预处理导致（125）I-表皮生长因子结合短暂但显著减少。竞争性结合实验表明，结合减少主要是由于亲和力降低，而不是表皮生长因子结合位点数量的变化。生长激素对表皮生长因子结合的影响具有浓度依赖性，并且在时间上与生长激素诱导的ERK激活和表皮生长因子受体PTP101反应性磷酸化相关。阻断MEK1/ERK途径而非蛋白激酶C途径可阻止生长激素对表皮生长因子结合的影响，我们的结果表明，生长激素和佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导的表皮生长因子结合抑制机制有很大不同。总体而言，我们的研究结果表明，生长激素可以通过MEK1/ERK途径依赖性方式调节表皮生长因子结合动力学和表皮生长因子受体结合后信号传导行程。
