Stoichiometry and modular retrieval of ENaC

ENaC 的化学计量和模块化检索

基本信息

批准号：
7390345
负责人：
James D Stockand
金额：
$ 22.78万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7390345
关键词：
Address Affect Architecture Biochemical Biochemistry Biology Blood Pressure Cell membrane Chinese Hamster Ovary Cell Clathrin Consensus Sequence Dominant-Negative Mutation Dynamin Dynamin 2 Electrophysiology (science)Endocytosis Epithelial Figs - dietary Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Half-Life Hydration status Image Ion Channel MAP Kinase Gene Measurement Mediating Membrane Methodology Mutation Outcome Pathway interactions Phosphorylation Physiological Play Principal Investigator Production Proteins Rate Regulation Relative (related person)Research Retrieval Role Route Signal Transduction Surface Testing Time Tyrosine Ubiquitination adaptor protein complex 2, mu 2 subunit base coated pit epithelial Na+ channel epsin experience fluorophore insight interest membrane activity mutant programs reconstitution research study stoichiometry ubiquitin ligase

Address Affect Architecture Biochemical Biochemistry Biology Blood Pressure Cell membrane Chinese Hamster Ovary Cell Clathrin Consensus Sequence Dominant-Negative Mutation Dynamin Dynamin 2 Electrophysiology (science)Endocytosis Epithelial Figs - dietary Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Half-Life Hydration status Image Ion Channel MAP Kinase Gene Measurement Mediating Membrane Methodology Mutation Outcome Pathway interactions Phosphorylation Physiological Play Principal Investigator Production Proteins Rate Regulation Relative (related person)Research Retrieval Role Route Signal Transduction Surface Testing Time Tyrosine Ubiquitination adaptor protein complex 2, mu 2 subunit base coated pit epithelial Na+ channel epsin experience fluorophore insight interest membrane activity mutant programs reconstitution research study stoichiometry ubiquitin ligase

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项目摘要

The epithelial Na channel (ENaC) plays a fundamental role in establishing blood pressure. ENaC activity is set, in part, by its level in the plasma membrane. Membrane levels of ENaC reflect constitutive delivery and regulated retrieval. The mechanisms and domains within ENaC involved in retrieval are not completely understood. We are interested in two conserved, overlying domains (S/TPPPxYxS/TL) found within all ENaC subunits: the PY (xPPxY) and tyrosine-based endocytic (YxxL) motifs. The prior motif targets the channel for ubiquitinylation via Nedd4 ubiquitin ligases; and the latter motif is known to interact with the mu2 subunit of the AP-2 complex, which targets proteins for clathrin coated-pit endocytosis mediated by dynamin. We will test whether these domains are modular and thus, separable or whether they act in concert. Physiological signaling cascades (e.g. MAPK) decrease ENaC activity by promoting channel degradation. Thus, we also test the hypothesis that MAPK signaling decreases channel activity via these modular retrieval domains. Moreover, we will determine whether absolutely conserved S/T just preceding the PY and within the YxxL motifs, which fit the consensus sequence for MAPK, are targets for MAPK impacting channel activity and membrane level. ENaC is a heteromeric channel comprised of 3 distinct subunits with channels containing two or fewer types of subunits having decreased activity. Thus, one possible outcome of subunit retrieval is production of homomeric channels or channels containing only two types of subunits. Since, subunit stoichiometry of membrane ENaC may not be fixed, we also ask whether MAPK signaling via the PY and YxxL domains changes ENaC subunit stoichiometry and/or composition to modulate channel activity. Here, we address four specific aims: 1) Determine subunit stoichiometry of membrane ENaC; 2) Determine whether membrane ENaC levels are controlled by modular PY and YxxL endocytic domains and assign significance to each domain; 3) Determine whether physiological cell signaling cascades modulate channel retrieval via the PY and YxxL motifs and whether differential phosphorylation of conserved S/T modulate this retrieval; and 4) Determine if retrieval of ENaC subunits from the plasma membrane is coordinated. This research will provide critical insight about the molecualr architecture of ENaC and regulation of this important channel.

上皮NA通道（ENAC）在建立血压方面起着基本作用。 ENAC活动是部分设置为质膜中的水平。 ENAC的膜水平反映了本构的递送和监管检索。检索中涉及的ENAC内的机制和域并不完全理解。我们对所有内部发现的两个保守的，上覆的域（s/tpppxyxs/tl）感兴趣 ENAC亚基：PY（XPPXY）和基于酪氨酸的内吞（YXXL）基序。先前的主题目标通过NEDD4泛素连接酶进行泛素化的通道；已知后者与MU2相互作用 AP-2复合物的亚基，该亚基靶向由dynamin介导的蛋白质涂层涂层的内吞作用。我们将测试这些域是否是模块化的，因此可以分离或它们是否共同起作用。生理信号传导级联（例如MAPK）通过促进通道降解来降低ENAC活性。因此，我们还测试了以下假设：MAPK信号通过这些模块化检索降低通道活动域。此外，我们将确定在PY之前和内部是否绝对保守的S/T 符合MAPK共识序列的YXXL图案是MAPK撞击通道的目标活动和膜水平。 ENAC是一个由3个不同的亚基组成的杂体通道包含两种或更少类型的亚基，其活性降低。因此，亚基的可能结果检索是仅包含两种类型亚基的同源通道或通道的生产。自从，膜ENAC的亚基化学计量法可能无法固定，我们还询问MAPK信号是否通过PY传导和YXXL域更改ENAC亚基化学计量和/或组成，以调节通道活动。在这里，我们解决了四个具体目标：1）确定膜ENAC的亚基化学计量； 2）确定膜ENAC水平是否由模块化PY和YXXL内吞域控制并分配对每个领域的意义； 3）确定生理细胞信号是否调制通道通过PY和YXXL基序检索，以及保守的S/T的差异磷酸化是否会调节这一点检索； 4）确定从质膜中检索ENAC亚基是否是协调的。这研究将提供有关ENAC的分子结构和对这一重要的调节的批判性见解渠道。