REGULATION OF APOPTOSIS BY THE SEROTONIN 1A RECEPTOR

5-羟色胺 1A 受体对细胞凋亡的调节

• 批准号: 2612538
• 负责人: PROBAL BANERJEE
• 金额: $ 7.71万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2612538
• 关键词: SDS polyacrylamide gel electrophoresis; aging; antibody; apoptosis; autoradiography; biological signal transduction; cell growth regulation; chemical association; cysteine endopeptidases; cytochrome c; enzyme activity; enzyme inhibitors; immunoprecipitation; intermolecular interaction; laboratory mouse; mitogen activated protein kinase; neurons; phosphorylation; radiotracer; receptor binding; receptor expression; serotonin receptor; spectrin; tissue /cell culture; western blottings

Aging is concomitant with progressive loss of neurons mainly through the slow, suicidal process of apoptosis. While the resulting loss of physical and cognitive functions have been studies by many groups, only sparse information is available on receptor-mediated, neuroprotective signaling cascades which undergo a significant decline during aging. Earlier studies have shown that the serotonin 1A receptor (5-HT1A-R) undergoes an age-related decline, however, the possibility that this loss of 5-HT1A-R expression could be a reason for neuronal apoptosis has not been addressed. Preliminary data show that the agonist-bound 5- HT1A-R causes inhibition of apoptosis by stimulating the mitogen activated kinase (MAPK) via intermediate proteins Ras and Raf-1. However apoptosis has been directly linked to increased Cytochrome c release from mitochondria, proteolytic activation of the cytosolic, interleukin 1beta-convertase (ICE)-like protease CPP32 and subsequent breakdown of cytoskeletal proteins (e.g. Fodrin) and nuclear DNA. The relationship between receptor mediated activation of MAPK and the pathways leading to CPP32 processing and activation is not known. This pilot project is designed to yield information which will form the foundation for a broader study to link these two events. Preliminary data showing 5-HT1A-R mediated inhibition of apoptosis, CPP32 processing and Fodrin cleavage in neuronal cells suggest that agonist binding to the 5-HT1A-R may cause an activation of the mitochondrial protein Bcl-2 which inhibits Cytochrome c release from mitochondria. This Bcl-2 activation could be through phosphorylation mediated inactivation of the death agonist protein, Bad, which before phosphorylation, binds to and inactivates Bcl-2. Serine-phosphorylation of Bad could be the result of 5-HT1A-R mediated activation of the serine/threonine kinase MAPK. Alternatively, the Bcl-2 binding protein Raf-1, upon MAPK mediated serine-phosphorylation and detachment from the plasma membrane, could bind to and transport the phosphoserine-binding, cytosolic protein 14-3-3 to the Bcl-2: Bad complex located on the mitochondria. Subsequent to its binding to Bcl-2, Raf-1 could cause serine- phosphorylation of Bad and prepare it for association with 14-3-3 and removal from Bcl-2, thus restoring Bcl-2 activity. For either model, 5-HT1A-R mediated increase in phosphorylation of Bad and association of phosphorylated Bad with 14-3-3 will be tested by 32PO43-labeling, immunoprecipitation and immunoblot analysis. MAPK mediated, direct phosphorylation of Bad will be tested through a time course analysis, since 5-HT1A-R mediated activation of MAPK occurs rapidly within 3 min. An indirect mechanism via phosphorylation of Raf-1, binding of phospho- Raf-1 to the Bcl-2:Bad complex and increase in phosphorylation of Bad should be a slower process requiring several protein-protein associations. Increased Raf-1 phosphorylation and Raf-1:Bcl-2 binding upon agonist activation of the 5-HT1A-R will be tested by 32PO43- labeling, coimmunoprecipitation of Raf-1 with Bcl-2 using Bcl-2 antibody and immunoblotting. The model confirmed will form the basis for future studies to elucidate the complete pathway from 5-HT1A-R activation to neuroprotection.

衰老与神经元的进行性丧失主要是通过 凋亡的缓慢，自杀过程。 而由此产生的损失 身体和认知功能是许多小组的研究，仅 稀疏信息可用于受体介导的神经保护 信号级联反应在衰老期间大幅下降。 较早的研究表明，5-羟色胺1A受体（5-HT1A-R） 然而，经历了与年龄有关的下降，这是 5-HT1A-R表达的丧失可能是神经元细胞凋亡的原因 没有解决。 初步数据表明，激动剂结合5- HT1A-R通过刺激有丝分裂来引起凋亡的抑制 激活的激酶（MAPK）通过中间蛋白Ras和RAF-1。 但是，细胞凋亡与细胞色素c的增加有关 从线粒体释放，胞质的蛋白水解活化 白介素1beta-convertase（ICE）类似蛋白酶CPP32，然后 细胞骨架蛋白（例如禽蛋白）和核DNA的分解。 这 受体介导的MAPK激活与 导致CPP32处理和激活的途径尚不清楚。 这 试点项目旨在产生将形成的信息 基金会进行了更广泛的研究，以将这两个事件联系起来。 初步的 数据显示5-HT1A-R介导的凋亡抑制，CPP32处理 神经元细胞中的禽蛋白裂解表明激动剂与 5-HT1A-R可能引起线粒体蛋白Bcl-2的激活 抑制线粒体中的细胞色素c释放。 这个Bcl-2 激活可能是通过磷酸化介导的 死亡激动剂蛋白质，不良，在磷酸化之前，与和 灭活Bcl-2。 不良的丝氨酸磷酸化可能是结果 5-HT1A-R介导的丝氨酸/苏氨酸激酶MAPK的激活。 或者，在MAPK介导 丝氨酸磷酸化和质膜脱离，可以 结合并运输磷酸糖结合，胞质蛋白 14-3-3 to Bcl-2：位于线粒体上的不良复合物。 在与Bcl-2结合后，RAF-1可能引起丝氨酸 - 不良磷酸化，并准备与14-3-3和 从Bcl-2中删除，从而恢复Bcl-2活性。 对于任何一个模型， 5-HT1A-R介导的不良磷酸化和关联的磷酸化增加 磷酸化的坏处和14-3-3将通过32PO43标签测试， 免疫沉淀和免疫印迹分析。 MAPK介导，直接 不良的磷酸化将通过时间课程分析测试， 由于5-HT1A-R介导的MAPK激活在3分钟内迅速发生。 通过RAF-1磷酸化的间接机制，磷酸的结合 RAF-1到BCL-2：不良复合物和不良磷酸化的增加 应该是需要几种蛋白质蛋白的过程较慢 协会。 RAF-1磷酸化增加和RAF-1：Bcl-2结合 5-HT1A-R的激动剂激活后，将通过32PO43-测试 标记，使用BCl-2抗体将RAF-1与BCL-2共免疫沉淀 和免疫印迹。 确认的模型将构成未来的基础 研究阐明从5-HT1A-R激活到的完整途径 神经保护。