Regulation of heterotrimeric G protein signaling by subunit phosphorylation

通过亚基磷酸化调节异源三聚体 G 蛋白信号传导

• 批准号: 10387072
• 负责人: SARAH M ASSMANN
• 金额: $ 7.75万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387072
• 关键词: Arabidopsis; Award; BODIPY; Biochemical; Biological Assay; Biology; Chemistry; Clinical; Communicable Diseases; Data; Development; Disease; Disease susceptibility; Eukaryota; Event; Funding; G-Protein Signaling Pathway; GTP Binding; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Genetic Diseases; Guanosine Triphosphate; Health; Heterotrimeric GTP-Binding Proteins; Human; Human Characteristics; Hydrolysis; In Vitro; Intervention; Laboratories; Luciferases; Measurement; Mediating; Methods; Molecular Probes; Mutation; Parents; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plant Model; Plants; Protein Subunits; Proteins; Reader; Regulation; Repression; Research; Research Personnel; Signal Transduction; Signaling Protein; Speed; System; United States National Institutes of Health; Validation; Variant; Work; data acquisition; design; fluorophore; instrumentation; mutant; protein protein interaction; small molecule; synergism; Arabidopsis; Award; BODIPY; Biochemical; Biological Assay; Biology; Chemistry; Clinical; Communicable Diseases; Data; Development; Disease; Disease susceptibility; Eukaryota; Event; Funding; G-Protein Signaling Pathway; GTP Binding; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Genetic Diseases; Guanosine Triphosphate; Health; Heterotrimeric GTP-Binding Proteins; Human; Human Characteristics; Hydrolysis; In Vitro; Intervention; Laboratories; Luciferases; Measurement; Mediating; Methods; Molecular Probes; Mutation; Parents; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plant Model; Plants; Protein Subunits; Proteins; Reader; Regulation; Repression; Research; Research Personnel; Signal Transduction; Signaling Protein; Speed; System; United States National Institutes of Health; Validation; Variant; Work; data acquisition; design; fluorophore; instrumentation; mutant; protein protein interaction; small molecule; synergism

Summary Heterotrimeric G protein signaling pathways are of tremendous importance to human health. Mutation of G protein subunits causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. Indeed, G protein pathways are the targets of approximately a third of all drugs under clinical use. The Assmann laboratory has furthered fundamental understanding of heterotrimeric G protein signaling through elucidation of G protein-mediated signaling cascades and phenotypes. The emphasis of the parent award is on mechanisms of phospho-regulation of G protein signaling that are evolutionarily conserved but have been oft overlooked in mammalian systems. In this research, the model plant Arabidopsis is used as a facile system to investigate kinase-mediated phosphorylation of the canonical G protein α (Gα) subunit, GPA1, and to explicate the downstream signaling impacts of this phosphorylation; in particular, how phosphorylation biases interactions with downstream effector proteins. In parallel, relevance to human health is demonstrated through assessment of the impacts of analogous phosphorylation events on human Gα subunits in vitro, using BODIPY-GTP binding and hydrolysis assays on the corresponding human phosphomimic mutants. This supplement requests funding for the purchase of a BioTek Synergy Neo2 plate reader to increase the throughput and reliability of the BODIPY- GTP activity assays and to allow the implementation of orthogonal methods, particularly transcreener assays, that will allow independent validation of these biochemical data. The dual monochromators of the Synergy Neo2 plate reader will allow for assay versatility with a multitude of fluorophores. The speed and sensitivity of the Synergy Neo2 far outpaces that of the extant obsolete Flx800 plate reader, allowing for finer timescale measurements of a greater number of Gα variants. In addition, the capability of the Synergy Neo2 to assay luciferase activity will facilitate assessment of biased signaling arising from phosphorylation-dependent protein- protein interactions, as the Synergy Neo2 has the capability for high-throughput measurements of protein-protein interaction using the split-luciferase method. In summary, the proposed state-of-the-art plate reader will provide reliable, sensitive, and rapid data acquisition as well as entirely new capabilities to probe the molecular effects of G protein phosphorylation. Finally, the instrumentation will be available to other NIH-funded researchers at Penn State in the Biology and Chemistry Departments.

概括 异三聚体G蛋白信号通路对人类健康至关重要。 g的突变 蛋白质亚基引起遗传疾病，发育异常和感染性疾病的改变 敏感性。实际上，G蛋白途径是临床使用中所有药物的大约三分之一的靶标。 Assmann实验室通过 阐明G蛋白介导的信号级联和表型。父母奖的重点是 G蛋白信号传导的磷酸调节机制，在进化上保守但有许多 在哺乳动物系统中被忽略。在这项研究中，模型植物拟南芥被用作一个轻松的系统 研究了典型G蛋白α（Gα）亚基，GPA1的激酶介导的磷酸化，并解释 该磷酸化的下游信号传导影响；特别是，磷酸化如何偏向相互作用 带有下游效应蛋白。同时，通过评估证明了与人类健康的相关性 使用Bodipy-GTP结合，类似磷酸化事件对人类Gα亚基的影响 并在相应的人磷酸突变体上进行水解测定。此补充要求资金 为了购买Biotek Synergy Neo2读取器，以提高Bodipy-的吞吐量和可靠性 GTP活动评估，并允许实施正交方法，尤其是经屏幕测定法， 这将允许对这些生化数据独立验证。 Synergy Neo2的双重单色器 板读取器将允许使用多种荧光团的测定能力。速度和灵敏度 Synergy Neo2远远超过了额外的过时FLX800读取器的空间，允许更精细的时间尺度 大量Gα变体的测量。此外，协同NEO2的能力分析能力 荧光素酶活性将促进评估由磷酸化依赖性蛋白质引起的偏置信号传导 蛋白质相互作用，因为协同NEO2具有高通量测量的能力 使用裂开酸酶方法相互作用。总而言之，拟议的最新板读取器将提供 可靠，敏感和快速的数据获取以及探测分子效应的全新功能 G蛋白磷酸化。最后，该仪器将向其他NIH资助的研究人员提供 宾夕法尼亚州生物学和化学部门。