ADAMs: Key regulators of EGFR signaling

描述（由申请人提供）：蛋白水解已成为表皮生长因子受体（EGFR）配体的关键翻译后调节因子，EGFR是一种在发育和疾病（如癌症）中具有重要作用的酪氨酸激酶受体。所有EGFR配体都是作为膜锚定的前体制备的，其胞外域经常需要蛋白水解释放或“脱落”以触发EGFR信号传导。ADAM（去整合素和金属蛋白酶）蛋白家族的金属蛋白酶在脱落六种EGFR配体中具有关键作用，并且缺乏ADAM 17的小鼠类似于缺乏EGFR的动物，或缺乏ADAM 17底物TGF α、HB-EGF和双调蛋白的动物。这项研究的主要目的是阐明亚当斯在脱落和激活EGFR配体中的关键作用的机制。具体而言，我们将：1）进行结构/功能分析以了解亚当斯10和17的底物选择性和调节。我们将在亚当斯10和17之间以及在ADAM 10底物EGF和ADAM 17底物TGF α之间产生嵌合体，以鉴定这些酶和底物的哪些结构域是其底物选择性所需的。此外，我们将确定细胞内信号传导途径的不同激活剂和抑制剂如何影响亚当斯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，因为它需要两个单独的配体结合事件之前，被占用的受体可以二聚化。预计未切割的膜束缚配体会在低配体浓度下阻碍受体二聚化，这可能解释了为什么这些配体的切割对于某些条件下的近分泌信号传导（细胞-细胞信号传导）至关重要。然而，配体的聚集或过表达可能允许受体二聚化，从而即使当它们不被切割时也允许阿曲他克林信号传导。我们将通过评估低浓度或高浓度的不可切割的TGF α，或由四跨膜蛋白CD 9或二聚化的化学诱导剂聚集的TGF α如何影响EGFR信号传导来检验这一假设。3)使用ADAM 17条件性敲除小鼠与乳腺中表达不同水平TGF α的转基因小鼠杂交，研究目的2结果的体内相关性。我们预计，拟议的研究将提供令人兴奋的新见解EGFR途径的上游调控蛋白水解的配体。由于EGFR信号传导在癌症等疾病中起着至关重要的作用，我们希望这项工作将为设计能够影响EGFR信号传导的药物发现新的靶点。

DESCRIPTION (provided by applicant): 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 of ADAMs lO and 17. We will generate chimera between ADAMs lO 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 ADAMs lO 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.