Molecular Biology of 5HT2A receptor expressing synapses

描述(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方面发挥着核心作用。5HT2A激动剂产生精神分裂效应，而5HT2A拮抗剂似乎有助于提高非典型抗精神病药物的疗效。因此，5HT2A受体活性的失调可能与精神分裂症和/或抑郁症有关。5HT2a受体表达于大脑对认知和情绪控制至关重要的区域的突触中，即大脑皮质的V层和整个边缘系统。这一建议涉及在分子和细胞水平上研究表达突触的5HT2A受体的结构和功能。为了发现与5HT2A型受体相互作用的新型脑蛋白，在酵母双杂交检测系统中，5HT2A型受体的c-末端尾巴被用作“诱饵”。三个编码人脑蛋白质区域的“阳性”克隆被发现与5HT2a受体c末端尾巴有很强的相互作用。经GenBank同源检索，阳性克隆编码“PDZ结构域”蛋白的部分序列。这项建议涉及使用酵母双杂交分析系统来发现和研究与5HT2A受体相互作用的新蛋白的性质，并可能参与含5HT2A受体的突触的结构和功能。主要目的是：L)利用我们建立的酵母双杂交方法鉴定人脑中与人5HT2A受体C末端的相互作用蛋白质；2)从我们酵母双杂交文库鉴定的部分克隆中获得全长克隆；3)使用GST-融合蛋白实验验证酵母双杂交实验中候选蛋白质在酵母细胞外的相互作用；4)使用免疫共沉淀实验研究重组细胞系中全长5HT2A受体与候选蛋白质的相互作用。这些研究旨在为R01的应用提供基础，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，可能包括可能在精神疾病中被破坏的受体调节机制。

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.