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

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.

上皮钠通道 (ENaC) 在建立血压方面发挥着重要作用。 ENaC 活性为部分由其在质膜中的水平决定。 ENaC 的膜水平反映了组成型递送和受监管的检索。 ENaC 中涉及检索的机制和领域并不完全明白了。我们对在所有结构域中发现的两个保守的重叠结构域 (S/TPPPxYxS/TL) 感兴趣。 ENaC 亚基：PY (xPPxY) 和基于酪氨酸的内吞 (YxxL) 基序。先前的主题针对的是通过 Nedd4 泛素连接酶进行泛素化的通道；已知后一个基序与 mu2 相互作用 AP-2 复合物的亚基，其目标蛋白是由动力介导的网格蛋白包被的凹坑内吞作用。我们将测试这些域是否是模块化的，因此是可分离的，或者它们是否协同作用。生理信号级联（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 MAPK 信号传导通过这些模块化检索降低通道活动的假设域。此外，我们将确定 PY 之前和内部的 S/T 是否绝对保守 YxxL 基序符合 MAPK 的共有序列，是 MAPK 影响通道的目标活性和膜水平。 ENaC 是一个异聚通道，由 3 个不同的亚基组成，通道含有两种或更少类型的活性降低的亚基。因此，亚基的一种可能结果修复是同聚通道或仅包含两种类型亚基的通道的产生。自从，膜 ENaC 的亚基化学计量可能不固定，我们还询问 MAPK 信号是否通过 PY 和 YxxL 结构域改变 ENaC 亚基化学计量和/或组成以调节通道活性。在这里，我们解决四个具体目标：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 内吞结构域控制并分配对每个领域的重要性； 3) 确定生理细胞信号级联是否调节通道通过 PY 和 YxxL 基序进行检索以及保守 S/T 的差异磷酸化是否对此进行调节检索； 4) 确定从质膜中恢复 ENaC 亚基是否协调。这研究将为 ENaC 的分子结构和这一重要的调控提供重要见解渠道。