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.

在过去的一年里，我们描述并扩展了我们新的原位 受体-G-蛋白测定系统，以提高其实用性 不同的受体结构家族。视网膜激活 脱辅基视蛋白已被检查为配体的分子模型 受调节的 G 蛋白偶联受体；以及之间的关系 G-蛋白α和β-γ亚基与视网膜的相互作用和 测试了视紫红质构象。这些实验表明，正如所见 对于真正的配体调节受体，apo-的视网膜激活 视蛋白显示两种亲和状态。不过亲和力高 视蛋白与视网膜的相互作用似乎受β-控制 γ而不是α亚基相互作用。作为 我们利用表面进行 G 蛋白偶联的视紫红质模型 用于 G- 动力学分析的等离子共振 (SPR) 测量 实时监测蛋白质-视紫红质相互作用。功能上 活性视紫红质通过结合固定在 SPR 表面 固定化刀豆球蛋白A.以这种方式固定化的视紫红质 保留了催化的全部生化比活性 视网膜G蛋白α亚基上的核苷酸交换。这 视网膜α和β-γ亚基的结合相互作用 视紫红质具有深刻的协同作用。 β-γ二聚体的结合 与视紫红质不同的 γ 亚基，与 α 无关 亚基，很容易通过 SPR 观察到。此外，这些二聚体 显示出显着不同的结合亲和力和动力学。 生理上适当的视网膜二聚体表现出快速 缔合和解离动力学，而其他β-γ 二聚体解离速度慢 100 倍以上。这些数据 表明 G 蛋白偶联受体信号传导的持续时间 事件是 G 蛋白偶联伙伴的固有特性， 特别是β-γ二聚体。这些发现正在 随后分析 β-γ 的结合相互作用 具有嵌合伽马亚基链的二聚体和视紫红质。我们 启动了 G 蛋白信号传导的分子研究 人类大麻素受体。两个基因已被鉴定 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、抗呕吐和 通过 Gi 和 Go 信号传导进行免疫抑制，但是由于它们 广泛的影响和精神活性特性的治疗 目前可用的激动剂的效用是有限的。我们建造了 编码人类受体的杆状病毒并检查其配体 通过原位重建进行调节和 G 蛋白选择性。 虽然 CB1 和 CB2 受体均激活 Gi 和 Go， CB1 激活 Gi 或 Go 的功效是激动剂依赖性的。我们的 因此，数据证明了激动剂选择性 G 蛋白信号传导 CB1 受体并表明治疗剂可以设计为 选择性调节个体 G 蛋白信号通路。我们在 原位测定程序可以为合理设计提供工具 非致醉性大麻素制剂。我们继续我们的 铃蟾肽受体分子特性的研究 胃泌素释放肽家族突变分析 受体（GRP-R）。定点 GRP-R 突变体在 成纤维细胞并测定其结合配体的能力 催化鸟嘌呤核苷酸的交换。两个氨基酸位置 预计会形成分子内盐桥（D98 和 R309） 在大多数 G 蛋白偶联中发现了类似的潜在离子对 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-R 突变（D98R、R309D）不活跃。相比之下， 双交换突变（D98R/R309D）具有高亲和力激动剂结合， 并具有较高的内在基础活性和整体催化活性 与野生型相比的交换活性。这些发现是 与这两个极性之间的盐桥相互作用一致 和带相反电荷的氨基酸，保持适当的 与 G 蛋白相互作用所必需的受体构象。的 十个残基预测为潜在的配体结合接触点，两个 突变显示激动剂（Y285A）或拮抗剂选择性丧失 (F313A) 与 GRP-R 结合。此外，我们还鉴定出两个氨基 第六跨膜片段中的酸性残基 F270 和 N281 这对于受体构象变化至高水平至关重要 亲和力，催化活性状态。在竞争结合测定中 使用拮抗剂放射性配体，铃蟾肽显示出 20-100 倍 与 N281A 和 F270A 突变体 GRP-R 相比亲和力降低 至野生型受体。饱和结合等温线是最好的 通过双位点模型拟合，表明受体位于 低亲和力（KDL）或高亲和力（KDH）状态。的比例为 两种亲和力（KDL/KDH）均显着增加 突变体，而高亲和力突变受体的比例 状态 (RH/RTOTAL) 降低。我们得出结论，这两个残基 对于受体构象变化至高水平很重要 核苷酸有效催化所需的亲和状态 在Gq上兑换。