Molecular Biology of 5HT2A receptor expressing synapses

5HT2A 受体表达突触的分子生物学

• 批准号: 6869606
• 负责人: Milton Teitler
• 金额: $ 14.22万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-12 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869606
• 关键词: cell line; chimeric proteins; frontal lobe /cortex; gene mutation; glutathione transferase; immunoprecipitation; molecular cloning; polymerase chain reaction; protein protein interaction; protein structure function; receptor expression; serotonin receptor; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The 5HT2A receptor, a member of the G-protein-coupled receptor family (GPCR), appears to play a central role in controlling cognition and mood. 5HT2A agonists produce psychotomimetic effects and 5HT2A antagonism appears to contribute to the increased efficacy of atypical antipsychotic drugs. Therefore dysregulation of 5HT2A receptor activity may be involved in schizophrenia and/or depression. 5HT2A receptors are expressed in the synapses of brain regions critical for control of cognition and mood, i.e. layer V of the cortex and throughout the limbic system. This proposal involves the investigation of the structure and function of 5HT2A receptor expressing synapses on the molecular and cellular level. In order to discover novel brain proteins that interact with the 5HT2A receptor, the c-terminal tail of the 5HT2A receptor was used as the "bait" in a yeast two-hybrid assay system. Three "positive" clones, coding for regions of human brain proteins, were found to strongly interact with the 5HT2A receptor c-terminal tail. Based on homology searches of GENBANK, the positive clones code for partial sequences of "PDZ domain" proteins. This proposal involves the use of the yeast two-hybrid assay system to discover and investigate the properties of novel proteins that interact with the 5HT2A receptor and may participate in the structure and function of 5HT2A receptor-containing synapses. The specific aims are: l) to use the yeast two-hybrid assay we have developed to identify proteins in the adult human brain that can interact with the c-terminal tail of the human 5HT2A receptor; 2) to produce full-length clones from the partial clones identified from our yeast two-hybrid cDNA library; 3) to use the GST-fusion protein assay to validate the interaction of candidate proteins from the yeast two-hybrid assay outside of the yeast cell environment; 4) to use co-immunoprecipitation studies to demonstrate the interaction of the full-length 5HT2A receptor and candidate proteins in recombinant cell lines. These studies are designed to provide the foundation for an R01 application involving investigations of the structural and functional aspects of 5HT2A receptor synaptic molecular biology and may lead to the discovery of completely novel GPCR signal transduction mechanisms, possibly including receptor regulation mechanisms that may be disrupted in psychiatric disorders.

描述（由申请人提供）：5HT2A 受体是 G 蛋白偶联受体家族 (GPCR) 的成员，似乎在控制认知和情绪中发挥核心作用。 5HT2A 激动剂产生拟精神病作用，5HT2A 拮抗作用似乎有助于提高非典型抗精神病药物的疗效。因此，5HT2A 受体活性失调可能与精神分裂症和/或抑郁症有关。 5HT2A 受体在对控制认知和情绪至关重要的大脑区域的突触中表达，即皮质的 V 层和整个边缘系统。该提案涉及在分子和细胞水平上研究表达5HT2A受体的突触的结构和功能。为了发现与5HT2A受体相互作用的新型脑蛋白，5HT2A受体的C端尾部被用作酵母双杂交测定系统中的“诱饵”。三个“阳性”克隆，编码人脑蛋白区域，被发现与 5HT2A 受体 c 末端尾部强烈相互作用。基于GENBANK的同源性搜索，阳性克隆编码“PDZ结构域”蛋白的部分序列。该提案涉及使用酵母双杂交测定系统来发现和研究与 5HT2A 受体相互作用并可能参与含有 5HT2A 受体的突触的结构和功能的新型蛋白质的特性。具体目标是： l) 使用我们开发的酵母双杂交测定法来鉴定成人大脑中可以与人类 5HT2A 受体 C 末端尾部相互作用的蛋白质； 2) 从我们的酵母二杂交cDNA文库中鉴定出的部分克隆产生全长克隆； 3) 使用GST-融合蛋白测定来验证酵母细胞环境外酵母双杂交测定中候选蛋白的相互作用； 4) 使用免疫共沉淀研究来证明全长5HT2A受体与重组细胞系中候选蛋白的相互作用。这些研究旨在为 R01 应用提供基础，涉及 5HT2A 受体突触分子生物学的结构和功能方面的研究，并可能导致发现全新的 GPCR 信号转导机制，可能包括在精神疾病。