ADAMs: Key regulators of EGFR signaling

蛋白水解已成为表皮生长因子配体的关键翻译后调节因子 受体（EGFR），一种酪氨酸激酶受体，在发育和疾病中具有重要作用， 癌所有EGFR配体都是作为膜锚定的前体制备的，其胞外域通常 需要蛋白水解释放或“脱落”来触发EGFR信号传导。ADAM金属蛋白酶（a 去整合素和金属蛋白酶）蛋白家族在脱落六种EGFR配体中起关键作用， 缺乏ADAM 17的动物类似于缺乏EGFR的动物，或缺乏ADAM 17底物TGF α的动物， HB-EGF和双调蛋白。这项研究的主要目的是阐明 这是亚当斯在脱落和激活EGFR配体中的关键作用的基础。具体而言，我们将： 1)进行结构/功能分析，以了解底物选择性和ADAMslO的调节 和17.我们将在ADAM 10和17之间以及在ADAM 10底物之间产生嵌合体。 EGF和ADAM 17底物TGF α，以确定这些酶和底物的哪些结构域是 这是其底物选择性所必需的。此外，我们将建立不同的激活剂和抑制剂， 细胞内信号传导途径影响ADAM 10和17的功能。 2)研究ADAM 17在阿曲他克林信号传导中的作用。通过EGFR的信号传导是不寻常的，因为它需要两个 在被占据的受体可以二聚化之前分离配体结合事件。未裂膜栓系 预计配体在低配体浓度下阻碍受体二聚化，这可能解释了为什么 在某些条件下，这些配体的裂解对于阿曲他克林信号传导（细胞-细胞信号传导）是关键的。 然而，配体的聚集或过度表达可能会使受体二聚化，从而使阿曲他克林 即使它们没有被切割也发出信号。我们将通过评估低或高 浓度的不可裂解的TGF α，或由四跨膜蛋白CD 9或由化学物质聚集的TGF α 二聚化的诱导剂，影响EGFR信号传导。 3)使用ADAM 17交叉的条件性敲除小鼠解决目标2结果的体内相关性 用在乳腺中表达不同水平TGF α的转基因小鼠。 我们预计，拟议的研究将提供令人兴奋的新见解的上游调控， EGFR途径通过其配体的蛋白水解。因为EGFR信号在疾病中起着至关重要的作用， 癌症，我们希望这项工作将揭示新的目标，设计药物，可以影响EGFR信号。

Proteolysis has emerged as a key posttranslational regulator of ligands of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with important roles in development and diseases such as cancer. All EGFR-ligands are made as membrane anchored precursors whose ectodomains frequently require proteolytic release or "shedding" to trigger EGFR-signaling. Metalloproteases of the ADAM (a disintegrin and metalloprotease) protein family have key roles in shedding six EGFR-ligands, and mice lacking ADAM17 resemble animals lacking the EGFR, or animals lacking the ADAM17 substrates TGFa, HB-EGF and amphiregulin. The main goal of the proposed research is to elucidate the mechanism underlying the critical role of ADAMs in shedding and activating ligands of the EGFR. Specifically, we will: 1) Perform a structure/function analysis to understand the substrate selectivity and regulation ofADAMslO and 17. We will generate chimera between ADAMslO and 17 as well as between the ADAM10 substrate EGF and the ADAM17 substrate TGFa to identify which domains of these enzymes and substrates are required for their substrate selectivity. Moreover, we will establish how different activators and inhibitors of intracellular signaling pathways affect the function of ADAMslO and 17. 2) Study the role ofADAM17 in juxtacrine signaling. Signaling via the EGFR is unusual in that it requires two separate ligand binding events before the occupied receptors can dimerize. Uncleaved membrane tethered ligands are predicted to impede receptor dimerization at low ligand concentrations, which might explain why cleavage of these ligands is critical for juxtacrine signaling (cell-cell signaling) under certain conditions. However, clustering or overexpression of ligands might allow receptor dimerization and thus juxtacrine signaling even when they are not cleaved. We will test this hypothesis by assessing how low or high concentrations of uncleavable TGFa, or of TGFa that is clustered by the tetraspanin CD9 or by chemical inducers of dimerization, affect EGFR-signaling. 3) Address the in vivo relevance of the results of aim 2 using conditional knockout mice forADAM17 crossed with transgenic mice expressing different levels of TGFa in the mammary gland. We anticipate that the proposed studies will provide exciting new insights into the upstream regulation of the EGFR pathway by proteolysis of its ligands. Because EGFR-signaling has a crucial role in diseases such as cancer, we hope this work will uncover new targets for the design of drugs that can affect EGFR signaling.