Biogenesis of the Regulated Secretory Pathway

调节分泌途径的生物发生

基本信息

批准号：
7675565
负责人：
Daniel W. Sirkis
金额：
$ 3.2万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-16 至 2013-04-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7675565
关键词：
Adaptor Signaling Protein Behavior Behavioral Biochemical Biogenesis Biological Assay Cell membrane Cell surface Cells Chromogranins Cytoplasm Dense Core Vesicle Density Gradient Centrifugation Disease Double-Stranded RNA Drosophila genus Endocytosis Event Exocytosis Flow Cytometry Foundations Future Genes Glutamates Golgi Apparatus Growth Factor Hand Immunofluorescence Immunologic Inorganic Sulfates Libraries Mammalian Cell Mammals Mediating Membrane Membrane Proteins Molecular Monitor Mutagenesis Mutation Neuroendocrine Cell Neuromodulator Neurosecretory Systems Neurotransmitters Opioid Organelles PC12 Cells Pain Pathway interactions Peptides Phenocopy Physiological Process Protein Secretion Proteins RNA Interference Rattus Relative (related person)Reporter Rewards Role Schizophrenia Screening procedure Secretory Vesicles Signal Transduction Sorting - Cell Movement System Unspecified or Sulfate Ion Sulfates Vesicle Work base depression in vivo knock-down luminal membrane monoamine mutant neuropsychiatry neurotrophic factor peptide hormone protein transport relating to nervous system research study response secretory protein trafficking uptake vesicular monoamine transporter

项目摘要

DESCRIPTION (provided by applicant): Neuromodulators including monoamine neurotransmitters and neural peptides control a wide range of behavioral phenomena, and their dysregulation contributes to neuropsychiatric disease, but we still understand little about the cellular mechanisms that underlie their regulated release. In particular, we do not understand how proteins sort to the large dense core vesicles (LDCVs) that mediate regulated release, rather than to the vesicles that mediate constitutive secretion. Previous work has even suggested a role for lumenal and membrane rather than cytoplasmic interactions of the kind that contribute to other trafficking events such as endocytosis. To identify factors involved in the formation of LDCVs, I am using the vesicular monoamine transporter (VMAT2), a polytopic membrane protein which fills secretory vesicles with monoamine neurotransmitters for exocytotic release. VMAT2 sorts efficiently and directly into the regulated secretory pathway (RSP), and mutations in a conserved, cytoplasmic dileucine-like motif divert VMAT2 to the constitutive pathway, increasing cell surface delivery. This motif appears important for sorting VMAT2 directly into LDCVs, suggesting that, in contrast to previous work, cytoplasmic factors mediate the sorting of VMAT2to the RSP and thus contribute to the biogenesis of LDCVs. We aim to identify the cellular machinery involved in formation of the RSP, using an unbiased RNAi screen in Drosophila S2 cells. Further, we will characterize the role of identified cellular factors in mammalian PC12 cells. In particular, we will determine whether these factors are responsible specifically for the sorting of VMAT2,or more generally for biogenesis of the pathway. Our studies will enable us to identify cellular factors involved in formation of the regulated secretory pathway. This information will allow us to manipulate regulated and constitutive secretion and determine their relative role in normal behavior and psychiatric illness

描述（由申请人提供）：包括单胺神经递质和神经肽在内的神经调节剂控制着广泛的行为现象，它们的失调及其失调导致神经精神病，但我们仍然对细胞机制几乎没有理解，而细胞机制构成了受调节释放的基础。特别是，我们不了解蛋白质如何分类介导调节释放的大型密集的核心囊泡（LDCV），而不是介导介导本构分泌的囊泡。先前的工作甚至提出了腔和膜的作用，而不是造成其他运输事件（例如内吞作用）的那种胞质相互作用。为了识别LDCV形成的因素，我使用的是囊泡单胺转运蛋白（VMAT2），这是一种多重膜蛋白，用单胺神经递质填充分泌囊泡以进行外生胞毒性释放。 VMAT2有效地直接分类为受调节的分泌途径（RSP），并在保守的，细胞质Dilecine样基序中的突变将VMAT2转移到组成途径，从而增加了细胞表面递送。该基序对于将VMAT2直接分类到LDCV中似乎很重要，这表明与先前的工作相反，细胞质因子介导VMAT2的分类为RSP，从而有助于LDCV的生物发生。我们旨在使用果蝇S2细胞中的无偏RNAi筛选来确定与RSP形成有关的细胞机制。此外，我们将表征已鉴定的细胞因子在哺乳动物PC12细胞中的作用。特别是，我们将确定这些因素是针对VMAT2的分选还是更普遍的途径生物发生负责。我们的研究将使我们能够确定与受调节分泌途径形成的细胞因素。这些信息将使我们能够操纵受监管和构成分泌，并确定它们在正常行为和精神病中的相对作用