Regulated cell surface Pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process
Regulated cell surface Pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process
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DOI：
10.1074/jbc.m307745200
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发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Mauduit, P
中科院分区：
文献类型：
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作者：
Le Gall, SM;Auger, R;Mauduit, P
表皮生长因子受体（EGFR）配体作为暴露在细胞表面的I型膜蛋白前体被合成。这些蛋白质的外结构域脱落是细胞调节这些生长因子的细胞相关形式和扩散形式之间平衡的方式。虽然转化生长因子- α、HB-EGF和双调节蛋白前体的调节脱落已被明确确立，但全长促egf脱落的调节尚未得到明确证明。在这里，我们使用野生型和M2突变型CHO-K1以及HeLa细胞系瞬时转染表位标记的大鼠促EGF表达质粒，证明这些细胞合成EGF作为在细胞表面表达的高分子量膜相关前体糖蛋白。一旦前体细胞出现在细胞表面，所有细胞系都能够在细胞外培养基中随着前体细胞近膜分裂释放整个前表皮生长因子的外畴。更重要的是，我们清楚地确定CHO-M2和HeLa细胞仅组成性地释放低水平的促egf。这种脱落在野生型CHO细胞中是一种受调节的现象，它可以被不同的药物如12-肉豆蔻酸13-醋酸酯（PMA）、高钒酸盐和血清诱导，但不能被钙离子载体诱导。使用特异性抑制剂以及蛋白激酶C （PKC）耗竭，PMA刺激被证明完全依赖于PKC的激活，而pervanadate和血清刺激则不是。调节的外胞结构域脱落涉及锌金属蛋白酶的活性，这是通过菲安特罗林和TAPI-2的抑制以及CHO-M2脱落缺陷突变细胞系的结果确定的。CHO和HeLa细胞系在刺激下释放促egf和促tnf - α的能力的比较极大地表明，TACE （adam17）可能不是参与促egf外域分泌的外蛋白酶，该蛋白酶仍有待鉴定，其细胞表达模式受到限制。
Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.