STRUCTURE AND FUNCTIONS OF SIGNAL TRANSDUCING G-PROTEINS

信号转导 G 蛋白的结构和功能

• 批准号: 2578781
• 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/2578781
• 关键词: Baculoviridae; G protein; biological signal transduction; bombesin; conformation; isoprenoid; neuropeptide receptor; protein reconstitution; protein structure function; receptor binding; receptor coupling; receptor expression; rhodopsin; serotonin receptor; transfection /expression vector

This past year we have continued our research efforts to develop appropriate in vitro techniques for the examination of G-protein-receptor interactions at the molecular level. This includes adapting our baculoviral expression vectors for simplified isolation of recombinant expressed 5HT2c receptors; modifying our chaotropic salt and G-protein reconstitution procedures for bombesin (GRP) receptors expressed in BalbC fibroblast; and improving the immobilization chemistries for coupling G- protein subunits and rhodopsin for surface plasmon resonance studies of protein-protein interaction. In addition, we have completed a detailed characterization of the molecular bases for "inverse" agonism of antagonist of the 5HT2c receptor expressed in insect Sf9 cells using the baculovirus system (Hartman & Northup, J. Biol. Chem. In press) and an initial characterization of the GRP receptor expressed in BalbC fibroblasts (Hellmich, Battey & Northup, submitted). We have as well continued to exploit visual transduction models for a detailed examination of the molecular interactions of G-protein subunits, receptor and guanine nucleotides. These studies have led us to propose that the catalytic function of receptors in G-protein activation involves an ordered interaction in which Gbeta-gamma binding to receptor proceeds Galpha-GDP and the product of the catalysis is a dissociated G`[empty] (Hoon, Clark, Ryba & Northup, manuscript in preparation). Prior models of G-protein activation have not ordered subunit interaction and have hypothesized that receptors stabilize the Galpha[empty] requiring GTP binding to dissociate the activated Galpha-GTP. Further, we have devised strategies of nucleotide competition which allow us to examine the receptor-catalysis function in some detail. While these experiments were designed to establish optimal kinetic protocols for testing models of G- protein-receptor interaction, they have provided as a by-product an enormously improved procedure for assay of selective G-protein-receptor interaction which promises to allow refined examination of these processes at the level of cellular membrane factions (Clark, Gutierrez & Northup, manuscript in preparation). Further, these data confirm by independent methods the conclusions we have reached using immobilized rhodopsins and measuring G-protein interactions by surface plasmon resonance (Clark, Mitchell, Chen & Northup, manuscript in preparation).

在过去的一年中，我们继续进行研究以发展 适合检查G蛋白受体的体外技术 分子水平的相互作用。 这包括适应我们的 细菌病毒表达载体，用于简化重组分离 表达5HT2C受体；修改我们的混沌盐和G蛋白 BALBC表达的孟买（GRP）受体的重建程序 成纤维细胞；并改善用于耦合g-的固定化学成分 用于表面等离子体共振研究的蛋白质亚基和视紫红质研究 蛋白质蛋白质相互作用。 此外，我们已经完成了详细的 分子碱基的表征，用于“反向”激动 使用昆虫SF9细胞表达的5HT2C受体的拮抗剂 杆状病毒系统（Hartman＆Northup，J。Biol。Chem。印刷中）和 BALBC中表达的GRP受体的初始表征 成纤维细胞（Hellmich，Battey＆Northup，提交）。 我们也有 继续利用视觉转导模型进行详细 检查G蛋白亚基，受体的分子相互作用 和鸟嘌呤核苷酸。 这些研究使我们提出 G蛋白激活中受体的催化功能涉及 GBETA-GAMMA与受体的结合的有序相互作用 Galpha-GDP和催化的产物是分解的G` [空] （Hoon，Clark，Ryba＆Northup，手稿准备）。 先前的模型 G蛋白的激活未订购亚基相互作用，并且具有 假设受体稳定需要GTP的Galpha [空] 结合以解离激活的Galpha-GTP。 此外，我们设计了 核苷酸竞争的策略使我们能够检查 受体催化功能详细介绍。 这些实验是 旨在建立最佳的动力学方案，用于测试G-的模型 蛋白质受体相互作用，它们作为副产品A提供了 大大改进了选择性G蛋白受体的方法 有望允许对这些的互动 细胞膜派系水平的过程（Clark，Gutierrez ＆Northup，准备手稿）。 此外，这些数据通过 独立方法我们使用固定的结论得出的结论 Rhodopsins和表面等离子体测量G蛋白相互作用 共鸣（Clark，Mitchell，Chen＆Northup，准备中的手稿）。