SEROTONIN BINDING PROTEIN--FUNCTION IN SYNAPTIC VESICLES

血清素结合蛋白——突触小泡的功能

• 批准号: 3376214
• 负责人: HADASSAH TAMIR
• 金额: $ 10万
• 依托单位: NEW YORK STATE PSYCHIATRIC INSTITUTE DBA RESEARCH FOUNDATION FOR MENTAL HYGIENE, INC
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3376214
• 关键词: affinity labeling; antiidiotype antibody; autoradiography; axon; brain; electrofocusing; electron microscopy; enzyme linked immunosorbent assay; gel electrophoresis; immunochemistry; ion exchange chromatography; monoclonal antibody; nerve endings; neurochemistry; neurons; neuropharmacology; neurotransmitter metabolism; nutrition related tag; organelles; phosphorylation; radiotracer; serotonin; synapses

The major objective of this proposal is to define the cellular and subcellular mechanism involved in the neuronal storage and release of serotonin (5-HT). Central and peripheral serotonergic neurons will be studied and in addition, a neurectodermally-derived paraneuron (the parafollicular cell of the thyroid) will also be examined. These three cell types all contain an intravesicular protein serotonin binding protein (SBP), that binds 5-HT with a high affinity under intracellular conditions. This protein is not found in 5-HT storing cells that are not of neurectodermal origin. Two forms of SBP, differing in molecular weight (45kDa and 56kDa) have been purified and characterized. Both are present in all 3 cell types although evidence suggests that the 56kDa form may be more prevalent in cell bodies and preterminal axons, while the 45kDa form appears to be more concentrated in nerve terminals. We propose that the 56kDa form of SBP which can be phosphorylated, is the precursor of the 45kDa material which cannot. In addition to testing this hypothesis, we will investigate the functional significance of the inhibition of 5-HT binding by SBP phosphorylation and the regulation of phosphorylation by Ca+2. In order to do this, the intracellular Ca+2 concentration will be manipulated pharmacologically and monitored with Quin-2. A photoaffinity probe, ANPA-5-HT, will be used to covalently label SBP either inside axon terminals or on their surface. This will be done to examine the possibility that SBP is retained by neurons when synaptic vesicles release their transmitter by exocytosis and is recycled. Maps of tryptic digests of the 45kDa and 56kDa forms of SBP will be constructed in order to examine the structural homologies between them. Monoclonal and polyclonal antibodies to SBP (as well as anti-idiotypic antibodies to 5-HT, which may react with SBP and 5-HT receptors) will be obtained and used both to study properties of the molecules and to explore the cellular and subcellular localization of SBP. Co-localization of 5-HT and SBP is predicted. Finally, the ontogency of 5-HT and SBP will be examined in relation to the appearance of synaptic vesicles. These studies of the brain and peripheral model systems should provide insights into critical elements of the cellular biology of serotonergic neurons that in turn should help in understanding those clinical conditions in which serotonergic function may be abnormal. In addition these studies should provide information necessary for designing pharmacological agents to manipulate serotonergic mechanisms.

该提案的主要目的是定义细胞和 涉及神经元存储和释放的亚细胞机制 5-羟色胺（5-HT）。 中央和周围血清素能神经元将是 研究和此外，神经抑制了衍生的paraneuron（ 还将检查甲状腺的副细胞）。 这三个 细胞类型均包含内内蛋白5-羟色胺结合蛋白 （SBP），在细胞内结合5-HT具有高亲和力 状况。 该蛋白在5-HT存储细胞中找不到 神经皮质的起源。 两种形式的SBP，分子量不同 （45KDA和56KDA）已被纯化和表征。 两者都在场 在所有3种细胞类型中，尽管有证据表明56KDA形式可能是 在细胞体和早产轴突中更普遍，而45KDA形成 似乎更集中在神经末端。 我们建议 56KDA形式的SBP形式可以被磷酸化，是 45KDA材料不能。 除了检验这一假设外，我们 将研究抑制5-HT的功能意义 通过SBP磷酸化结合和通过调节磷酸化的结合 CA+2。 为此，细胞内Ca+2浓度将是 通过药理操作并用Quin-2进行监测。 一个照片亲和力 探针ANPA-5-HT将用于共价标记Axon内部的SBP 终端或表面。 这将是为了检查 突触囊泡释放时神经元保留SBP的可能性 它们的发射器通过胞吐作用并回收。 胰蛋白酶摘要的地图 为了检查，将构建45KDA和56KDA形式的SBP 它们之间的结构同源。 单克隆和多克隆 SBP的抗体（以及5-HT的抗IDiotypic抗体，可能 将获得与SBP和5-HT受体的反应），并用于研究 分子的特性并探索细胞和亚细胞 SBP的本地化。 预测5-HT和SBP的共定位。 最后，将检查5-HT和SBP的成体性 突触囊泡的外观。 这些对大脑和周围的研究 模型系统应提供有关关键要素的见解 血清素能神经元的细胞生物学反过来有助于 了解5-羟色胺功能功能的临床条件 异常。 另外，这些研究应提供信息 设计药理学剂以操纵血清素能 机制。