STRUCTURE AND FUNCTION OF SIGNAL-TRANSDUCING G-PROTEINS

信号转导 G 蛋白的结构和功能

• 批准号: 3759497
• 负责人: J NORTHUP
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3759497
• 关键词: Baculoviridae; G protein; Sf9 cell line; biological signal transduction; in situ hybridization; laboratory rat; protein structure function; recombinant DNA; rhodopsin; serotonin; serotonin receptor

In this past year we have succeeded in a number of new initiatives designed to extend our understanding of the molecular interactions of G-proteins and receptors. First, we have confirmed the utility of the baculovirus insect cell system for the expression of functionally active recombinant receptors using rat 5HT2C receptor DNA. Analysis of the properties of the 5HT2C receptor by reconstitution in situ in Sf9 membranes has revealed a previously undescribed basal activity of the receptor and inverse agonist activity of several classical 5HT antagonist compounds. We have also developed procedures for removing the covalent retinal from bovine rhodopsin and subsequent activation of opsin by retinal analogs in order to exploit this as a model for ligand-operated receptors. Opsin also displays a basal activation which is suppressed by cis-retinals in the dark. Using the known structure of rhodopsins, we have modeled the retinal binding site of bovine rhodopsin and designed peptide structures as antagonists of retinal binding. This same model when applied to the 5HT2C receptor designed a distinct set of peptide structures which specifically occupy the 5HT binding site one of which was an agonist of the receptor. Lastly, we have succeeded in applying the novel biophysical approach of surface plasmon resonance to the examination of the protein-protein interactions of G-protein subunits and rhodopsin. The combination of recombinant expression of distinct molecular structures (and mutations) with in vitro biochemical and biophysical analyses should allow us to develop a molecular level description of receptor-G-protein coupling.

在过去的一年中，我们成功了许多新的 旨在扩展我们对 G蛋白和受体的分子相互作用。 第一的， 我们已经确认了杆状病毒昆虫的效用 用于表达功能活动的细胞系统 使用大鼠5HT2C受体DNA的重组受体。 分析5HT2C受体的性能 SF9膜的原位重构揭示了一个 以前未描述的受体的基础活性 几种经典5HT的反激动剂活性 拮抗剂化合物。 我们还开发了程序 用于从牛Rhopopsin和 随后通过视网膜类似物对Opsin激活OPSIN 将其作为配体锻炼受体的模型。 OPSIN还显示抑制的基础激活 由黑暗中的顺式视网膜通过。 使用已知的结构 Rhodopsins，我们已经建模了 牛Rhodopsin并将肽结构设计为 视网膜结合的拮抗剂。 当相同的模型 应用于5HT2C受体设计了一组独特的集合 专门占据5HT的肽结构 结合位点之一是受体的激动剂。 最后，我们成功地应用了新颖的生物物理 表面等离子体共振的方法 G蛋白亚基和 视紫红质。 重组表达的组合 具有体外的不同分子结构（和突变） 生化和生物物理分析应该使我们能够 发展受体-G蛋白的分子水平描述 耦合。