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

基本信息

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
项目类别：
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

项目摘要

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。蛋白质亚基导致遗传病、发育异常和传染病事实上，G 蛋白途径是临床使用的大约三分之一药物的靶标。 Assmann 实验室通过以下方法进一步加深了对异源三聚体 G 蛋白信号传导的基本了解阐明 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化调节机制在进化上是保守的，但已被经常研究在哺乳动物系统中被忽视的这一点在这项研究中，使用模式植物拟南芥作为一个简单的系统来解决这一问题。研究激酶介导的经典 G 蛋白 α (Gα) 亚基 GPA1 的磷酸化，并解释这种磷酸化的下游信号传导影响；特别是磷酸化如何影响相互作用；同时，通过评估证明与下游效应蛋白的相关性。使用 BODIPY-GTP 结合分析类似磷酸化事件对体外人 Gα 亚基的影响以及对相应的人类拟磷突变体进行水解测定本补充要求资助。购买 BioTek Synergy Neo2 读板机以提高 BODIPY- 的通量和可靠性 GTP 活性测定并允许实施正交方法，特别是转录筛选测定，这将允许对这些生化数据进行独立验证 Synergy Neo2 的双单色仪。酶标仪可实现多种荧光团的检测多功能性。酶标仪的速度和灵敏度。 Synergy Neo2 远远超过现有过时的 Flx800 读板器，可实现更精细的时间尺度此外，Synergy Neo2 还具有检测更多 Gα 变体的能力。荧光素酶活性将有助于评估磷酸化依赖性蛋白产生的偏向信号传导蛋白质相互作用，因为 Synergy Neo2 能够高通量测量蛋白质-蛋白质总之，所提出的最先进的读板器将提供使用分裂荧光素酶方法的相互作用。可靠、灵敏、快速的数据采集以及探测分子效应的全新功能最后，NIH 资助的其他研究人员将可以使用该仪器。宾夕法尼亚州立大学生物系和化学系。