ADAMs: Key Regulators of EGFR Signaling

描述（由申请人提供）：通过表皮生长因子受体（EGFR）的信号传导在发育和疾病（如癌症）中具有关键作用。EGFR的所有配体都被制成膜锚定的前体，其胞外域必须被蛋白水解释放或“脱落”以触发EGFR信号传导。因此，EGFR配体脱落对于EGFR信号传导至关重要。膜锚定金属蛋白酶ADAM 17已成为EGFR配体生物利用度的主要调节剂。缺乏ADAM 17的小鼠在表型上与缺乏EGFR的小鼠相似，为ADAM 17在EGFR信号传导中的重要作用提供了明确的遗传证据，并确立了ADAM 17作为EGFR依赖性癌症治疗的重要潜在靶点。ADAM 17活性受到高度调节，并受到许多信号通路的影响。这些途径如何在功能上与ADAM 17相交以及ADAM 17如何被激活是本申请的主题的关键问题。我们的结果表明，ADAM 17的突变体缺乏胞质结构域，因此没有潜在的磷酸化位点，对迄今为止测试的胞外域脱落的所有生理刺激反应正常。此外，从其他完整的膜蛋白的ADAM 17跨膜结构域（TMD）域取代废除ADAM 17激活。这指出了一种通过TMD调节ADAM 17的新机制。我们假设，ADAM 17 TMD整合并解释驱动ADAM激活的信号，并且是介导ADAM 17调节EGFR配体脱落的关键调节实体。 目的1是通过结构/功能研究确定TMD在激活ADAM 17中的作用。目的2的主要目标是通过将使用药理学化合物和siRNA技术的抑制实验与通过相关信号分子的强制表达激活信号通路相结合，来确定通过TMD激活ADAM 17的主要信号效应物。目的3中的实验将在缺乏ADAM 17胞质结构域的新敲入小鼠模型中严格测试ADAM 17胞质结构域在体内EGFR信号传导中的作用。 总之，这些目标将解决当前最紧迫的问题，即ADAM 17的调控，ADAM 17是一种主要的细胞脱落酶，是EGFR信号传导中的关键分子，也是癌症治疗的靶点。

DESCRIPTION (provided by applicant): Signaling through the epidermal growth factor receptor (EGFR) has critical roles in development and in diseases such as cancer. All ligands of the EGFR are made as membrane-anchored precursors whose ectodomain must be proteolytically released or "shed" to trigger EGFR-signaling. EGFR-ligand shedding is therefore crucial for EGFR- signaling. The membrane-anchored metalloproteinase ADAM17 has emerged as the principal regulator of the bioavailability of EGFR-ligands. Mice lacking ADAM17 phenotypically resemble those lacking the EGFR, providing unequivocal genetic evidence for the essential role of ADAM17 in EGFR signaling and establishing ADAM17 as an important potential target for the treatment of EGFR-dependent cancers. ADAM17 activity is highly regulated and is influenced by numerous signaling pathways. How these pathways functionally intersect with ADAM17 and how ADAM17 is activated are key questions that are the subject of this application. Our results show that a mutant of ADAM17 that lacks its cytoplasmic domain and therefore has no potential phosphorylation sites, responds normally to all physiological stimuli of ectodomain shedding tested to date. Moreover, substitution of the ADAM17 transmembrane domain (TMD) domain with that from other integral membrane proteins abolished ADAM17 activation. This points to a novel mechanism of ADAM17 regulation via its TMD. We hypothesize that the ADAM17 TMD integrates and interprets signals that drive ADAM activation and is the critical regulatory entity mediating the regulation of EGFR-ligand shedding by ADAM17. Aim 1 is focused on defining the role of the TMD in activating ADAM17 through structure/function studies. The main goal of aim 2 is to define the major signaling effectors that activate ADAM17 via its TMD by combining inhibition experiments using pharmacological compounds and siRNA technology with activation of signaling pathways by forced expression of relevant signaling molecules. The experiments in aim 3 will rigorously test the role of the ADAM17 cytoplasmic domain in EGFR signaling in vivo in a new knock-in mouse model lacking the ADAM17 cytoplasmic domain. Together, these aims will resolve the most pressing current questions regarding the regulation of ADAM17, a major cellular sheddase that is a critical molecule in EGFR signaling and a target for treatment of cancer.