SEROTONIN BINDING PROTEIN--FUNCTION INSYNAPTIC VESICLES

血清素结合蛋白--非突触小泡的功能

• 批准号: 3376215
• 负责人: HADASSAH TAMIR
• 金额: $ 12.29万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3376215
• 关键词: affinity labeling; binding proteins; brain; chemical binding; dogs; gel electrophoresis; genetic translation; immunoelectron microscopy; laboratory rabbit; laboratory rat; microsomes; molecular weight; monoclonal antibody; neurogenesis; neuropeptides; neurotransmitter metabolism; organelles; protein biosynthesis; protein purification; protein sequence; protein signal sequence; protein structure function; radiotracer; reticulocytes; serotonin; synaptic vesicles; tissue /cell culture; ubiquitin

The major objective of this proposal is to characterize cellular and subcellular mechanisms involved in the neuronal storage and release of serotonin (5-HT). Although found only in relatively small numbers of central and peripheral neurons, this neurotransmitter has major effect on behavior and mood. During the previous project period two specific serotonin (5-HT)) binding proteins (45 kDa and 56 kDa SBP) were purified and characterized. These proteins are found only in that subset of 5-HT-storing cells that is derived from neuroectoderm. It was proposed that 56 kDa SBP may be a precursor of the 45 kDa protein and that 45 kDa SBP may play a role in the storage of 5-HT in synaptic vesicles. These hypotheses will be tested in the proposed research (i) In order to determine if 56 kDa SBP is the physiological precursor of the 45 kDa molecule a determination will be made of (a) the extent of sequence homology between 56 kDa and 45 kDa SBP; (b) whether metabolically labeled 35S-56 kDa SBP is synthesized before 35S-45 kDa SBP and whether or not the 56 kDa protein is converted to the 45 kDa material. Monospecific antibodies to 45 kDa and 56 kDa SBP that were prepared during the prior project period will make these studies possible. This investigation will be facilitated by studying a recently characterized cell line (MTC cells) that produces and stores both 5-HT and SBP. If SBP is really an intravesicular 5-HT storage protein, it would be expected to be synthesized with a signal sequence, translated on polysomes attached to the RER, and cotranslationally segregated in the lumen of the RER. These predictions will be tested by in vitro translation using poly(A)+RNA extracted from the rat brain. The products (and their resistance to proteolysis) of in vitro translation with a rabbit reticulocyte lysate system will be compared with those synthesized on dog pancreas microsomes, in order to determine if cleavage of a signal peptide occurs and if there is vectorial transport of newly synthesized SBP into microsomes. If the initial experiments show that 56 kDa is produced before the 45 kDa SBP the intracellular compartment where the 56 kDa leads to 45 SBP conversion occurs, will be located. This will be done by following the fate, under control and experimental conditions, of pulse labeled SBP as it passes through successive subcellular compartments during a chase period. In additional experiments the ontogeny of serotonergic synapses will be studied. In particular, whether the timing of the appearance of 45 kDa SBP during ontogeny coincides with the development of serotonergic synaptic vesicles will be determined. Finally, the possibilities that 45 kDa SBP is co-released with 5-HT from activated serotonergic cells and is recaptured will be tested.

该提案的主要目的是表征蜂窝 以及与神经元存储有关的亚细胞机制和 释放5-羟色胺（5-HT）。 尽管仅在相对中发现 少数中央和周围神经元，这 神经递质对行为和情绪有重大影响。 在 上一个项目期间两个特定的5-羟色胺（5-HT））结合 纯化并表征蛋白质（45 kDa和56 kDa SBP）。 这些蛋白仅在5-HT存储细胞的该子集中发现 这是从神经外胚层得出的。 有人提出56 kDa SBP可能是45 kDa蛋白的前体，该蛋白质是45 kDa SBP 可能在突触囊泡中的5-HT中发挥作用。 这些 假设将在拟议的研究（i）中进行检验，以便 确定56 kDa SBP是否是45的生理前体 KDA分子将确定（a） 56 kDa至45 kDa SBP之间的序列同源性； （b）是否 代谢标记为35S-56 kDa SBP在35S-45之前合成 KDA SBP以及56 kDa蛋白是否转化为 45 kDa材料。 45 kDa和56 kDa SBP的单特异性抗体 是在上一期期间准备的 可能的研究。 这项调查将由 研究最近表征的细胞系（MTC细胞） 生产和存储5-HT和SBP。 如果SBP确实是 内部5-HT储存蛋白，预计将是 用信号序列合成，翻译在多个 附着在RER上，并在管腔中旋转分离 rer。 这些预测将通过体外测试 使用从大鼠脑提取的poly（a）+RNA的翻译。 这 体外产品（及其对蛋白水解的抗性） 带有兔子网状裂解液系统的翻译将是 与狗胰腺微粒体合成的那些相比 为了确定是否发生信号肽的切割以及是否发生 新合成的SBP矢量运输到 微粒体。 如果最初的实验表明56 kDa为 在45 kDa sbp之前生产的细胞内室，其中 56 kDa会导致45个SBP转换。 这将通过遵循命运，控制和 实验条件，脉搏标记为SBP时的实验条件 在追逐时期的连续亚细胞隔室。 在 其他实验血清素能突触的个体发育将 被研究。 特别是外观的时机 个体发育期间45 kDa SBP的发展与发展 将确定血清素能突触囊泡。 最后， 45 kDa SBP与5-HT共同发行的可能性 活化的血清素能细胞和重新接收。