ADAMs: Key Regulators of EGFR Signaling

描述(申请人提供)：通过表皮生长因子受体(EGFR)的信号在发育和癌症等疾病中起着关键作用。EGFR的所有配体都是作为膜锚定的前体，其胞外结构域必须被蛋白水解性释放或“脱落”以触发EGFR信号转导。因此，EGFR配体的脱落对EGFR信号转导至关重要。膜锚定的金属蛋白酶ADAM17已成为EGFR配体生物利用度的主要调节因子。缺乏ADAM17表型的小鼠与缺乏EGFR的小鼠相似，为ADAM17在EGFR信号转导中的重要作用提供了明确的遗传学证据，并确立了ADAM17作为治疗EGFR依赖癌症的重要潜在靶点。ADAM17的活性受到高度调控，并受到众多信号通路的影响。这些通路如何在功能上与ADAM17相交，以及ADAM17如何被激活是本应用程序的主题。我们的结果表明，ADAM17的突变体缺乏细胞质结构域，因此没有潜在的磷酸化位点，对迄今测试的所有胞外结构域脱落的生理刺激都能正常反应。此外，ADAM17跨膜区(TMD)结构域被其他完整膜蛋白取代后，ADAM17的激活被取消。这表明了一种通过其TMD调节ADAM17的新机制。我们推测，ADAM17 TMD整合并解释了驱动ADAM激活的信号，是介导ADAM17调节EGFR配体脱落的关键调控实体。目标1的重点是通过结构/功能研究确定TMD在激活ADAM17中的作用。目标2的主要目标是确定通过TMD激活ADAM17的主要信号效应器，方法是结合使用药理化合物和siRNA技术的抑制实验，以及通过强制表达相关信号分子来激活信号通路。Aim 3中的实验将严格测试ADAM17胞浆结构域在体内EGFR信号转导中的作用，在缺乏ADAM17胞质结构域的新敲入小鼠模型中。总之，这些目标将解决目前关于ADAM17调控的最紧迫的问题，ADAM17是一种主要的细胞脱落酶，是EGFR信号转导中的关键分子，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.