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Molecular regulation of the AP2 clathrin adaptor complex

AP2 网格蛋白接头复合物的分子调控

基本信息

批准号：
10582196
负责人：
Gunther Hollopeter
金额：
$ 24.99万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10582196
关键词：
Address Alzheimer&apos s Disease Animals Binding Caenorhabditis elegans Cardiovascular Diseases Cardiovascular system Cell membrane Cell physiology Cells Cholesterol Clathrin Clathrin Adaptors Complex Data Disease Ear Endocytosis Ensure Escape Mutant Genetic Screening Goals Growth Factor Health Heart Hepatitis In Vitro Influenza Ligands Malignant Neoplasms Mediating Medical Membrane Mitochondria Molecular Molecular Conformation Molecular Machines Mutagenesis Names Neurodegenerative Disorders Neuromodulator Pathologic Phospholipids Phosphorylation Phosphotransferases Physiological Process Protein Family Proteins Receptor Signaling Recycling Regulation Research Role Scanning Structure Techniques Testing Therapeutic Vesicle Virus Virus Diseases enhancer-binding protein AP-2 in vivo innovation macromolecule mutant neoplastic novel particle rational design receptor binding screening spatiotemporal tool

项目摘要

Abstract Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signaling receptors, this fundamental cellular process also tunes our sensitivity to the potentially pathological actions of growth factors and neuromodulators. As such, understanding how the underlying endocytic machinery is regulated promises to reveal novel mechanisms that could be harnessed to control neoplastic, neurodegenerative, cardiovascular, and viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin coat. Despite the central role of AP2, we lack critical details about how this molecular machine is regulated in vivo. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2 activity at multiple levels and have employed deep genetic screens to identify two conserved protein families that appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulators of the endocytic machinery function mechanistically. Previously it was thought that membrane phospholipids, cytosolic cargo domains, and phosphorylation by the AP2-associated kinase (AAK1) activate AP2. Our preliminary data indicate that a conserved region of the membrane-associated Fer/Cip4 Homology Domain-only (FCHo) proteins is required to promote endocytosis by converting AP2 to an active complex. We have named this functionally important domain the AP2 Activator, or APA. In Aim 1 we will test whether the APA is sufficient to induce a structural rearrangement of AP2, as well as defining the roles of membrane, cargo, and phosphorylation in that process. We will determine where the APA binds to AP2 by screening for C. elegans mutants that escape an APA anchored to mitochondria. We will evaluate the physiological significance of AP2 phosphorylation by characterizing kinase mutants. In Aim 2 we will validate our hypothesis that adaptiN-Ear-Binding Coat- Associated Proteins (NECAP)s counteract the active (open) conformation of AP2 to ensure proper recycling of adaptor complexes. We have discovered that AP2 accumulates in a hyper-open, hyper-phosphorylated state in NECAP mutants, and that NECAPs specifically bind open, phosphorylated forms of AP2. We will determine how NECAPs regulate AP2 activity and where they function within the hierarchy of AP2 modulation using in vitro and in vivo approaches. To fully understand how NECAPs function, we will determine their structure, and use an innovative random-scanning mutagenesis technique to determine the relevant NECAP-AP2 contacts in vivo. The long-term impact of the proposed research will be to clarify how fundamental cellular machinery is controlled with spatiotemporal precision in metazoans – where misregulation leads to important diseases.

摘要