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).

在过去的一年里，我们继续努力开发 用于检查 G 蛋白受体的适当体外技术 分子水平上的相互作用。 这包括调整我们的 用于简化重组分离的杆状病毒表达载体 表达5HT2c受体；修改我们的离液盐和 G 蛋白 BalbC 中表达的铃蟾肽 (GRP) 受体的重构程序 成纤维细胞；并改进偶联 G- 的固定化化学 蛋白质亚基和视紫红质用于表面等离子共振研究 蛋白质-蛋白质相互作用。 此外，我们还完成了详细的 “反向”激动的分子基础的表征 使用昆虫 Sf9 细胞中表达的 5HT2c 受体拮抗剂 杆状病毒系统（Hartman & Northup, J. Biol. Chem. In press）和 BalbC 中表达的 GRP 受体的初步表征 成纤维细胞（Hellmich、Battey 和 Northup，已提交）。 我们也有 继续利用视觉转导模型来详细了解 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 与受体结合的有序相互作用 Galpha-GDP 和催化产物是游离的 G`[空] （Hoon、Clark、Ryba 和 Northup，手稿正在准备中）。 先前型号 G 蛋白激活没有有序的亚基相互作用，并且具有 假设受体稳定需要 GTP 的 Galpha[empty] 结合以解离激活的 Galpha-GTP。 此外，我们还设计了 核苷酸竞争策略使我们能够检查 受体催化功能的一些细节。 虽然这些实验是 旨在建立最佳动力学协议来测试 G-模型 蛋白质-受体相互作用，它们作为副产品提供了 选择性 G 蛋白受体测定程序的极大改进 互动有望允许对这些进行精细的检查 细胞膜派系水平上的过程（Clark、Gutierrez 和诺瑟普，手稿正在准备中）。 此外，这些数据证实 独立方法我们使用固定化方法得出的结论 视紫红质并通过表面等离子体测量 G 蛋白相互作用 共振（Clark、Mitchell、Chen 和 Northup，手稿正在准备中）。