Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, Japan.
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka, Japan.
J Biol Chem. 2020 Aug 28;295(35):12343-12352. doi: 10.1074/jbc.RA120.013758. Epub 2020 Jun 24.
Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two ctivated ell dhesion olecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.
细胞外结构域脱落是一种翻译后修饰机制，通过这种机制，膜蛋白的整个细胞外结构域通过跨膜加工而释放。由于脱落会迅速且不可逆地改变细胞的特性，因此该过程受到适当的调节。然而，控制膜蛋白脱落倾向的分子机制在很大程度上尚不清楚。在这里，我们提供的证据表明，在脱落发生的无规卷曲跨膜区的茎区中带负电荷的氨基酸有助于脱落的易感性。我们表明，通过选择性剪接产生的两种激活的细胞黏附分子（ALCAM）蛋白变体对 ADAM 金属肽酶结构域 17（ADAM17）介导的脱落具有不同的易感性。值得注意的是，包含由 39 个碱基对长的选择性外显子编码的茎区赋予了脱落抗性。我们发现，这个选择性外显子编码了很大一部分带负电荷的氨基酸，我们证明这些氨基酸对于赋予脱落抗性是不可或缺的。我们还表明，将带负电荷的氨基酸引入到脱落敏感的 ALCAM 变体蛋白的茎区中可以减弱其脱落。此外，我们观察到，驻留在 Erb-B2 受体酪氨酸激酶 4（ERBB4）的茎区中的带负电荷的氨基酸对于其脱落抗性是不可或缺的。总的来说，我们的结果表明，茎区中的带负电荷的氨基酸会干扰多种膜蛋白的脱落。我们得出结论，茎区的组成决定了膜蛋白的脱落易感性。
