Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: Role of the intracellular adapter protein Fe65
Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: Role of the intracellular adapter protein Fe65
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DOI：
10.1523/jneurosci.21-21-08354.2001
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发表时间：
2001-11-01
影响因子：
5.3
通讯作者：
Hyman, BT
中科院分区：
文献类型：
--
作者：
Kinoshita, A;Whelan, CM;Hyman, BT
淀粉样蛋白-β是阿尔茨海默病中老年斑的主要成分，其通过蛋白水解衍生自淀粉样前体蛋白（APP）。含有Kunitz蛋白酶抑制剂（KPI）的APP（APP 751/770）与多功能内吞受体（低密度脂蛋白受体相关蛋白（LRP））相互作用，调节其影响淀粉样蛋白β产生的蛋白水解过程。我们用荧光共振能量转移（FRET）技术在H4细胞系中检测了标记的LRP和APP的亚细胞定位和参与APP-LRP相互作用的分子结构域。含KPI形式的APP（APP 770）显示与LRP的FRET对LRP抑制剂受体相关蛋白（RAP）敏感，表明APP 770和LRP的胞外结构域之间存在相互作用。APP 695也与LRP发生较低程度的相互作用（通过胞外结构域探针测量），并且这种胞外结构域相互作用不被RAP改变。通过使用C-末端标记的LRP和APP，我们证明了APP 695和APP 770的C末端与LRP的C末端之间的第二个相互作用位点，并且这些区域的相互作用对RAP不敏感。我们接下来检查了C-末端APP-LRP相互作用由Fe 65介导的可能性，Fe 65是与LRP和APP的胞质尾相互作用的衔接蛋白。FRET研究证实了氨基Fe 65磷酸酪氨酸结合（PTB）结构域和LRP胞质结构域之间以及羧基Fe 65 PTB结构域和APP胞质结构域之间的紧密接近。这些发现表明，LRP与APP的相互作用通过细胞外和细胞内蛋白质相互作用结构域发生。
Amyloid-beta, the major constituent of senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-beta. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.