Cell surface ectodomain cleavage of human amphiregulin precursor is sensitive to a metalloprotease inhibitor -: Release of a predominant N-glycosylated 43-kDa soluble form
Cell surface ectodomain cleavage of human amphiregulin precursor is sensitive to a metalloprotease inhibitor -: Release of a predominant N-glycosylated 43-kDa soluble form
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DOI：
10.1074/jbc.273.27.17258
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发表时间：
1998-07-03
影响因子：
4.8
通讯作者：
Dempsey, PJ
中科院分区：
文献类型：
--
作者：
Brown, CL;Meise, KS;Dempsey, PJ
已经在人结肠直肠癌（HCA-7，Caco-2）和乳腺癌（MCF-7）细胞系以及稳定表达各种人AR前体（pro-AR）形式的Madin-Darby犬肾细胞中研究了双调蛋白（AR）的生物合成和加工。表达内源性和转染AR的细胞均产生多种细胞和可溶性形式的AR，其中N-糖基化的50-kDa前AR形式占主导地位。我们的研究结果表明，连续的蛋白水解裂解的胞外域内的50 kDa的pro-AR形式导致释放的主要N-糖基化的43 kDa的可溶性AR，以及其他细胞和可溶性AR形式的外观。使用C-末端表位标记的pro-AR的细胞表面生物素化研究表明，所有细胞表面形式都是膜锚定的，并支持AR通过质膜上pro-AR的胞外域切割释放。我们还表明，前AR胞外域切割是一个受调节的过程，它可以刺激佛波醇12-肉豆蔻酸酯13-乙酸酯和金属蛋白酶抑制剂，巴马司他抑制。此外，我们提供的证据表明，高分子量AR形式可能保留全长N-末端前区，这可能会影响这些形式的生物活性。
Biosynthesis and processing of amphiregulin (AR) have been investigated in human colorectal (HCA-7, Caco-2) and mammary (MCF-7) cancer cell lines, as well as in Madin-Darby canine kidney cells stably expressing various human AR precursor (pro-AR) forms. Both cells expressing endogenous and transfected AR produce multiple cellular and soluble forms of AR with an N-glycosylated 50-kDa pro-AR form being predominant. Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominant N-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length N-terminal pro-region, which may influence the biological activities of these forms.