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.

在过去的一年里，我们取得了一系列新成果 旨在加深我们对 G 蛋白和受体的分子相互作用。 第一的， 我们已经证实了杆状病毒昆虫的效用 表达功能活性的细胞系统 使用大鼠 5HT2C 受体 DNA 重组受体。 5HT2C受体特性分析 Sf9 膜的原位重建揭示了 先前未描述的受体的基础活性和 几种经典5HT的反向激动剂活性 拮抗剂化合物。 我们还制定了程序 用于从牛视紫红质中去除共价视网膜 随后通过视网膜类似物激活视蛋白 利用它作为配体操作受体的模型。 视蛋白还显示出被抑制的基础激活 在黑暗中通过顺式视网膜。 使用已知的结构 视紫红质，我们模拟了视网膜结合位点 牛视紫红质并设计了肽结构 视网膜结合拮抗剂。 同一型号的时候 应用于 5HT2C 受体设计了一组独特的 特异性占据5HT的肽结构 结合位点，其中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学 表面等离子体共振检查的方法 G蛋白亚基的蛋白质-蛋白质相互作用和 视紫红质。 重组表达的组合 与体外不同的分子结构（和突变） 生化和生物物理分析应该使我们能够 开发受体-G-蛋白的分子水平描述 耦合。