Molecular Biology of 5HT2A receptor expressing synapses

描述（由申请人提供）：5HT2A受体是g蛋白偶联受体家族（GPCR）的一员，似乎在控制认知和情绪方面起着核心作用。5HT2A激动剂产生拟精神作用，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受体活性失调可能与精神分裂症和/或抑郁症有关。5HT2A受体在大脑中控制认知和情绪的关键区域的突触中表达，即皮层的V层和整个边缘系统。本研究拟从分子和细胞水平对5HT2A受体表达突触的结构和功能进行研究。为了发现与5HT2A受体相互作用的新脑蛋白，5HT2A受体的c端尾部被用作酵母双杂交试验系统的“诱饵”。三个“阳性”克隆，编码人类大脑蛋白质区域，被发现与5HT2A受体c端尾部强烈相互作用。根据GENBANK同源性检索，阳性克隆编码“PDZ结构域”蛋白的部分序列。本研究利用酵母双杂交实验系统发现和研究与5HT2A受体相互作用的新蛋白的特性，这些蛋白可能参与含有5HT2A受体的突触的结构和功能。具体目标是：1)使用我们开发的酵母双杂交试验来鉴定成人大脑中可以与人类5HT2A受体c端尾部相互作用的蛋白质；2)从酵母双杂交cDNA文库中鉴定的部分克隆获得全长克隆；3)利用gst融合蛋白实验验证酵母双杂交实验中候选蛋白在酵母细胞外的相互作用；4)利用共免疫沉淀研究证实全长5HT2A受体与候选蛋白在重组细胞系中的相互作用。这些研究旨在为涉及5HT2A受体突触分子生物学结构和功能方面研究的R01应用提供基础，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.