ADAMs: Key regulators of EGFR signaling

蛋白分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，在发育和疾病中发挥重要作用，如 癌症。所有的EGFR配体都被制成膜锚定的前体，其胞外结构域频繁 需要蛋白水解性释放或“脱落”来触发EGFR信号。亚当的金属蛋白酶(A) 去整合素和金属蛋白酶)蛋白家族在六种EGFR配体的脱落中起关键作用，小鼠 缺乏ADAM17的动物类似于缺乏EGFR的动物，或者缺乏ADAM17底物TGFa的动物， Hb-EGF和两用调节素。拟议的研究的主要目的是阐明这种机制。 亚当斯在脱落和激活EGFR配体方面的关键作用。具体来说，我们会： 1)进行结构/功能分析以了解ADAMsLO的底物选择性和调节 和17。我们将在ADAMs10和17之间以及ADAM10底物之间产生嵌合体 EGF和ADAM17底物TGFa，以确定这些酶和底物的哪些结构域 它们的底物选择性所需的。此外，我们将确定不同的激活剂和抑制剂是如何 细胞内信号通路影响ADAMslO和17的功能。 2)研究ADAM17在旁分泌信号中的作用。通过EGFR发送信号是不寻常的，因为它需要两个 在被占据的受体能够二聚化之前分离配体结合事件。无裂膜系留 在低配体浓度下，配体被预测会阻碍受体二聚化，这可能解释了为什么 在某些条件下，这些配体的切割对旁分泌信号(细胞-细胞信号)至关重要。 然而，配体的聚集或过度表达可能允许受体二聚化，从而导致伴随他克林 即使它们没有乳沟，也会发出信号。我们将通过评估低或高来检验这一假设 不可切割的TGFa的浓度，或由Tetraspanin CD9或通过化学物质聚集的TGFa的浓度 二聚化的诱导物，影响EGFR信号转导。 3)利用ADAM17杂交的条件基因敲除小鼠解决了AIM 2结果的体内相关性 转基因小鼠在乳腺中表达不同水平的TGFa。 我们预期拟议的研究将为上游监管提供令人振奋的新见解 EGFR途径通过其配体的蛋白分解。因为EGFR信号在疾病中起着至关重要的作用 对于癌症，我们希望这项工作将为可以影响EGFR信号转导的药物设计发现新的靶点。

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.