ANTIBODIES AGAINST PHOSPHORYLATED 5HT2C RECEPTORS

针对磷酸化 5HT2C 受体的抗体

• 批准号: 2848607
• 负责人: JON RANDALL BACKSTROM
• 金额: $ 7.6万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2848607
• 关键词: 3T3 cells; antibody; epitope mapping; gel mobility shift assay; gene deletion mutation; laboratory rabbit; laboratory rat; molecular site; phosphorylation; protein structure function; serotonin receptor; tissue /cell culture

Serotonin 5-HT2c receptors have been implicated in several neurologic conditions such as schizophrenia, overeating, depression, and epileptic- like seizures. However, it is unknown when and where 5-HT2C receptors function in the brain. Since 5-HT2C receptors are phosphorylated upon exposure to agonists, we will generate antibodies against phosphorylated epitopes to probe activation of 5-HT2C receptors in vivo. Specific aims are designed to: 1) identify phosphorylated sites of 5-HT2C receptors. Phospho-epitope mapping experiments will be performed with 5-HT2C receptor antibodies to identify potential phosphorylated regions of receptors expressed in NIH/3T3 cells. Deletion mutants lacking various regions of 5-HT2C receptors will be examined in gel-shift and phosphate-incorporation assays to identify phosphorylated regions. Wild-type receptors will be subjected to phosphoamino analyses to identity phosphorylated amino acids. Phospho-epitope mapping experiments will be performed with native receptors to test the hypothesis that native receptors are phosphorylated in the same region as recombinant receptors. The results from mutant receptors and phosphoamino analyses of wild-type receptors will be used to design site-directed mutants that lack potential phosphorylation sites. Incorporation of phosphate in the site-directed mutants will be measured to identify sites required for agonist-mediated phosphorylation of 5-HT2C receptors. 2) generate and characterize anti- (phospho)-peptide antibodies. Antibodies will be generated against phosphorylated peptides that correspond to the region(s) of 5-HT2C receptors sensitive to agonist-mediated phosphorylation. Once specificity of antibodies against the phosphorylated form of 5-HT2C receptors has been established in cell lines, reactivity of antibodies against native 5-HT2C receptors will be evaluated on immunoblots. The anti-(phospho)-peptide antibodies generated during this research period will be powerful tools to probe activation of 5-HT2C receptors in vivo. The long-term goal of this research is to address the role of 5-HT2C receptors in epilepsy and hallucinogenic drug activity.

5-羟色胺5-HT 2c受体与几种神经系统疾病如精神分裂症、暴食、抑郁和癫痫样发作有关. 然而，目前还不清楚5-HT 2C受体在大脑中何时何地发挥作用。 由于5-HT 2C受体在暴露于激动剂时被磷酸化，我们将产生针对磷酸化表位的抗体以探测体内5-HT 2C受体的激活。 具体目的设计为：1）鉴定5-HT 2C受体的磷酸化位点。 将使用5-HT 2C受体抗体进行磷酸化表位作图实验，以鉴定NIH/3 T3细胞中表达的受体的潜在磷酸化区域。缺失突变体缺乏5-HT 2C受体的各个区域将在凝胶位移和磷酸盐掺入试验中进行检查，以确定磷酸化区域。 将对野生型受体进行磷酸氨基分析以鉴定磷酸化氨基酸。 将使用天然受体进行磷酸化-表位作图实验，以检验天然受体在与重组受体相同的区域中磷酸化的假设。 野生型受体的突变受体和磷酸氨基分析的结果将用于设计缺乏潜在磷酸化位点的定点突变体。 将测量磷酸盐在定点突变体中的掺入，以鉴定激动剂介导的5-HT 2C受体磷酸化所需的位点。2)产生并表征抗（磷酸）肽抗体。 将产生针对磷酸化肽的抗体，所述磷酸化肽对应于对激动剂介导的磷酸化敏感的5-HT 2C受体的区域。 一旦在细胞系中确定了抗体对5-HT 2C受体磷酸化形式的特异性，将在免疫印迹中评价抗体对天然5-HT 2C受体的反应性。在此研究期间产生的抗（磷酸）肽抗体将是探测体内5-HT 2C受体激活的有力工具。 这项研究的长期目标是解决5-HT 2C受体在癫痫和致幻药物活性中的作用。