Physiology, Biophysics and Systems Biology Program, Weill Cornell Medicine, New York, New York, USA; Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, New York, USA.
Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, New York, USA.
J Biol Chem. 2021 Jan-Jun;296:100733. doi: 10.1016/j.jbc.2021.100733. Epub 2021 May 4.
A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17-/- mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.
解整合素金属蛋白酶 17（ADAM17）是一种细胞表面金属蛋白酶，作为肿瘤坏死因子 α（TNFα）、白细胞介素 6 受体（IL-6R）和表皮生长因子受体（EGFR）的几个配体的主要脱落酶，调节这些关键信号通路。ADAM17 的激活需要其跨膜结构域，但不需要细胞质结构域，而关于该结构域在体内的作用知之甚少。为了研究这一点，我们使用 CRISPR-Cas9 技术在小鼠中突变内源性 Adam17 基因座，产生缺乏细胞质结构域的突变 ADAM17（Adam17Δcyto）。纯合 Adam17Δcyto 动物以孟德尔比例出生，并存活至成年期，具有略微卷曲的毛发和卷曲的胡须，这与 ADAM17/EGFR 信号传导缺陷一致。在出生时，Adam17Δcyto 小鼠与 Adam17-/- 小鼠相似，它们的眼睛睁开，半月形心脏瓣膜增大，但它们没有骨生长板缺陷。细胞质结构域的缺失导致所有检查的组织和细胞中的 ADAM17 蛋白水平明显降低，这可能是低表型的原因。在功能测定中，Adam17Δcyto 小鼠胚胎成纤维细胞和骨髓来源的巨噬细胞的 ADAM17 活性明显降低，与蛋白水平降低一致。尽管如此，ADAM17Δcyto 可以被 PMA 刺激，PMA 是 ADAM17 的一种经过充分研究的翻译后激活剂，这证实了内源性 ADAM17 的细胞质结构域对于其对 PMA 的快速反应不是必需的。总之，这些结果首次提供了证据，证明 ADAM17 的细胞质结构域在体内在调节 ADAM17 水平和功能方面起着关键作用。
