Thorne B A, Plowman G D
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Mol Cell Biol. 1994 Mar;14(3):1635-46. doi: 10.1128/mcb.14.3.1635-1646.1994.
The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.
人表皮生长因子（EGF）家族的五个成员（EGF、转化生长因子α [TGF-α]、肝素结合表皮生长因子样生长因子 [HB-EGF]、双调蛋白和双向调节素 [AR]）最初被合成为跨膜蛋白，其细胞外结构域经过蛋白水解加工后释放出具有生物活性的成熟生长因子。这些因子都能激活表皮生长因子受体，但与EGF和TGF-α不同的是，HB-EGF和AR的成熟形式也是糖基化的肝素结合蛋白。我们构建了一系列突变体，以研究AR前体不同结构域在生物合成过程中的影响。前体的跨膜结构域和细胞质结构域对于从COS细胞或乳腺上皮来源的细胞中分泌生物活性AR并非必需，尽管在没有膜锚定的情况下，N端前区域的蛋白水解去除效率较低。然而，缺失N端前区域会导致该分子在细胞内迅速降解，没有可检测到的活性生长因子分泌。用HB-EGF前体的相应结构域取代天然前区域可保留AR的分泌，但用TGF-α前体的相应结构域则不行。在没有任何N端前区域的情况下，通过删除成熟AR的N端肝素结合结构域可恢复该分子的分泌。相比之下，EGF和TGF-α在没有其前区域的情况下也能分泌。然而，如果该蛋白与AR肝素结合结构域融合，除非同时存在AR前区域，否则TGF-α的分泌会受到抑制。我们提出，成熟AR的肝素结合结构域需要在N端前区域存在特定的结构基序，以允许生物活性分子正确折叠并因此分泌。
