Selective roles for tumor necrosis factor α-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family -: The juxtamembrane stalk determines cleavage efficiency
Selective roles for tumor necrosis factor α-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family -: The juxtamembrane stalk determines cleavage efficiency
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DOI：
10.1074/jbc.m312141200
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发表时间：
2004-06-04
影响因子：
4.8
通讯作者：
Lee, DC
中科院分区：
文献类型：
--
作者：
Hinkle, CL;Sunnarborg, SW;Lee, DC
表皮生长因子 (EGF) 家族配体是通过完整膜前体的胞外域的蛋白水解裂解而衍生的。此前，我们确定肿瘤坏死因子α转换酶（TACE/ADAM17）是一种生理性转化生长因子-α（TGF-α）脱落酶，并且我们还证明，在TACE缺陷的EC-2成纤维细胞中恢复TACE活性后，双调蛋白（AR）和肝素结合（HB）-EGF的脱落增强。在这里，我们通过证明纯化的可溶性 TACE 在体外切割小鼠 pro-HB-EGF 和 pro-AR 胞外域的近膜茎中的单个位点来扩展这些结果。对于 pro-HB-EGF，该位点与纯化的人生长因子的 C 末端相匹配，我们推测 AR 切割位点也具有生理相关性。相比之下，ADAM9和-10均与HB-EGF脱落有关，分别未能裂解胞外域或在非生理位点裂解。 EC-2 细胞中 TACE 的共转染增强了佛波醇肉豆蔻酸酯乙酸酯诱导的上皮调节蛋白的脱落，但不增强上皮调节蛋白的组成型脱落，并且对β细胞素 (BTC) 加工没有影响。此外，在体外，可溶性 TACE 不会裂解前 BTC 或前上皮调节蛋白胞外域的近膜茎。替换较短的原 BTC 近膜茎或截断原 TGF-α 茎以匹配原 BTC 长度可将转染细胞中的 TGF-α 脱落减少至背景水平，而替换原 BTC P2-P2' 序列则不太显着地减少 TGF-α 脱落。相反，TGF-α原茎的取代或BTC原茎的延长，特别是当与TGF-α原P2-P2'序列的取代相结合时，显着增加了BTC脱落。这些结果表明，有效的 TACE 裂解是由茎长度和易断裂键序列的组合决定的。
Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.