STRUCTURE AND FUNCTIONS OF SIGNAL-TRANSDUCING G-PROTEINS

信号转导 G 蛋白的结构和功能

• 批准号: 6289649
• 负责人: JOHN K NORTHUP
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 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/6289649
• 关键词: 3T3 cells; G protein; Sf9 cell line; allosteric site; biological signal transduction; bombesin; conformation; cyclic GMP; ketanserin; neuropeptide receptor; phosphodiesterases; piperazines; protein reconstitution; protein structure function; receptor coupling; receptor sensitivity; serotonin inhibitor; serotonin receptor; structural biology; visual perception

In the past year we have characterized and extended our new in situ receptor-G-protein assay systems to increase their utility for a diverse family of receptor structures. The retinal activation of apo-opsin has been examined as a molecular model for ligand- regulated G-protein coupled receptors; and the relationships between G-protein alpha and beta-gamma subunit and retinal interactions and rhodopsin conformations tested. These experiments show that, as seen for bona fide ligand-regulated receptors, retinal activation of apo- opsin displays two affinity states. However, the high affinity interaction of opsin with retinal appears to be controlled by beta- gamma rather than alpha subunit interaction. As an extension of the rhodopsin model for G protein coupling we have utilized surface plasmon resonance (SPR) measurements for kinetic analysis of G- protein-rhodopsin interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface via binding to immobilized concanavalin A. Rhodopsin immobilized in this manner retained full biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein alpha subunit. The binding interactions of retinal alpha and beta-gamma subunits with rhodopsin were profoundly synergistic. Binding of beta-gamma dimers with distinct gamma subunits to rhodopsin, independent of alpha subunit, was readily observable by SPR. Further, these dimers displayed dramatically different binding affinities and kinetics. The physiologically appropriate retinal dimer displayed rapid association and dissociation kinetics, while the other beta-gamma dimers dissociated at more than 100-fold slower rates. These data suggest that the duration of a G-protein-coupled receptor signaling event is an intrinsic property of the G protein coupling partners, in particular, the beta-gamma dimer. These findings are being followed up by analysis of the binding interactions of beta-gamma dimers and rhodopsin with chimeric gamma subunit chains. We initiated a molecular investigation of the G protein signaling of the human cannabinoid receptors. Two genes have been identified encoding receptors for cannabinoid ligands, CB1 and CB2. The CB1 and CB2 cannabinoid receptors mediate antinociception, antiemisis and immunosupression through Gi and Go signaling, however due to their wide range of effects and psychoactive properties the therapeutic utility of currently available agonists is limited. We constructed baculoviruses encoding the human receptors and examined their ligand regulation and G protein selectivity by in situ reconstitution. While the CB1 and CB2 receptors both activate Gi and Go, the efficacy of CB1 activation of Gi or Go is agonist-dependent. Our data thus demonstrate agonist-selective G-protein signaling by the CB1 receptor and suggest that therapeutic agents may be designed to regulate individual G-protein signaling pathways selectively. Our in situ assay procedures may provide a tool for the rational design of non-intoxicating cannabinoid agents. We have continued our investigations of the molecular properties of the bombesin receptor family by mutational analysis of the Gastrin Releasing Peptide Receptor (GRP-R). Site-directed GRP-R mutants were expressed in fibroblasts and assayed for their ability to bind ligands and to catalyze exchange of guanine nucleotides. Two amino acid positions were predicted to form an intramolecular salt-bridge (D98 and R309) and a similar potential ion pair is found in most G protein coupled receptors. Alanine substitution at these positions reduced agonist binding and G-protein activation compared to wild type GRP-R. Single swapGRP-Rmutations (D98R, R309D) wereinactive. In contrast, the double swap mutation (D98R/R309D) had high affinity agonist binding, and had both a higher intrinsic basal activity and overall catalytic exchange activity compared to wild type.Thesefindings are consistent with a saltbridge interaction between these two polar and oppositely charged amino acids that maintains the proper receptor conformation necessary to interact with G proteins. Of the ten residues predicted as potential ligand binding contacts, two mutations showed selective loss for agonist (Y285A) or antagonist (F313A) binding to the GRP-R. In addition, we identified two amino acid residues, F270 and N281, in the sixth transmembrane segment which are essential for the receptor conformational change to a high affinity, catalytically active state. In a competition binding assay using an antagonist radioligand, bombesin showed a 20-100 fold decreased affinity for the N281A and F270A mutant GRP-R as compared to the wild-type receptor. The saturation binding isotherms are best fit by a two-site model, indicating that the receptors are in either a low affinity (KDL) or a high affinity (KDH) state. The ratio of the two affinities (KDL/KDH) was significantly increased in both mutants, while the fraction of mutant receptors in the high affinity state (RH/RTOTAL) was decreased. We conclude that these two residues are important for receptor conformational change to the high affinity state required for efficient catalysis of nucleotide exchange on Gq.

在过去的一年中 受体-G蛋白测定系统，以增加其实用性 多元化的受体结构家族。视网膜激活 Apo-oppin已被检查为配体的分子模型 调节的G蛋白偶联受体；以及之间的关系 G蛋白alpha和beta-gamma亚基和视网膜相互作用以及 测试了视紫红质构象。这些实验表明，如所见 对于真正的配体调节的受体，apo-的视网膜激活 Opsin显示两个亲和力状态。但是，高亲和力 Opsin与视网膜的相互作用似乎是由β-控制的 伽玛而不是α亚基相互作用。作为扩展 G蛋白耦合的Rhodopsin模型我们已经使用了表面 血浆共振（SPR）测量用于G-的动力学分析 实时监测的蛋白质 - 偶像蛋白相互作用。在功能上 活性视紫红蛋白通过与 固定的姜黄素A.二红蛋白以这种方式固定 保留全生化特异性活性以催化 视网膜G蛋白α亚基上的核苷酸交换。这 视网膜α和β-gamma亚基的结合相互作用与 视紫红质是深刻的协同作用。 β-伽马二聚体的结合 与紫红素不同的伽马亚基，独立于α 亚基很容易被SPR观察到。此外，这些二聚体 显示出急剧不同的结合亲和力和动力学。 生理上适当的视网膜二聚体迅速显示 关联和解离动力学，而其他β-伽马 二聚体以速度慢100倍以上解离。这些数据 提示G蛋白偶联受体信号的持续时间 事件是G蛋白耦合伙伴的内在特性， 特别是β-gamma二聚体。这些发现正在 随后分析β-伽马的结合相互作用 带有嵌合伽玛亚基链的二聚体和视紫红质。我们 开始对G蛋白信号的分子研究 人类大麻素受体。已经鉴定出两个基因 编码大麻素配体CB1和CB2的受体。 CB1和 CB2大麻素受体介导抗伤害感受，抗炎和 通过gi进行免疫选择和发信号，但是由于它们 多种效果和精神活性特性治疗 当前可用激动剂的效用是有限的。我们构建了 编码人体并检查配体的杆状病毒 调节和G蛋白选择性通过原位重构。 虽然CB1和CB2受体都激活GI和GO，但 CB1激活GI或GO的功效依赖于激动剂。我们的 因此，数据证明了激动剂选择性G蛋白信号传导 CB1受体，并建议可以设计治疗剂 选择性调节单个G蛋白信号通路。我们进来 原位测定程序可以为合理设计提供工具 无毒性大麻素剂。我们继续我们的 研究孟买受体的分子特性 通过对胃释放肽的突变分析进行家庭分析 受体（GRP-R）。位于定点的GRP-R突变体在 成纤维细胞并测定其结合配体的能力和与 催化鸟嘌呤核苷酸的交换。两个氨基酸位置 预测形成分子内盐桥（D98和R309） 并且在大多数G蛋白偶联中都发现了类似的电势离子对 受体。这些位置的丙氨酸取代减少了激动剂 与野生型GRP-R相比，结合和G蛋白激活。单身的 SWAPGRP-RUMUTATION（D98R，R309D）具有性。相反， 双交换突变（D98R/R309D）具有高亲和力激动剂结合， 并且具有较高的固有活性和总体催化 与野生类型相比，交换活动是 与这两个极性之间的盐桥相互作用一致 并具有相反的电荷氨基酸，可维持适当的 与G蛋白相互作用所需的受体构象。的 十个残留物被预测为潜在的配体结合触点，两个 突变显示激动剂（Y285A）或拮抗剂的选择性损失 （F313a）与GRP-R结合。此外，我们确定了两个氨基 第六个跨膜段中的酸残基F270和N281 这对于受体构象变化至高度至关重要 亲和力，催化活性状态。在竞争绑定测定中 使用拮抗剂放射线，孟买显示出20-100倍 与N281A和F270A突变GRP-R相比，对N281A的亲和力降低了 到野生型受体。饱和绑定等温线是最好的 通过两个站点模型拟合，表明受体在任何一个 低亲和力（KDL）或高亲和力（KDH）状态。比率 这两个亲和力（KDL/KDH）都显着增加 突变体，而高亲和力中突变受体的比例 状态（rh/rtotal）减少。我们得出的结论是这两个残留物 对于对高的受体构象变化很重要 有效核苷酸的有效催化所需的亲和力状态 在GQ上进行交流。