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: 10379961
• 负责人: SARAH M ASSMANN
• 金额: $ 29.99万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379961
• 关键词: Address; Animals; Arabidopsis; Attention; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Cardiac health; Cells; Clinical; Communicable Diseases; Complement; Coupled; Coupling; Cyclic GMP-Dependent Protein Kinases; Data Set; Defect; Development; Disease; Disease susceptibility; Dissociation; Drug Targeting; Elements; Family; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Genetic; Genetic Diseases; Genetic Epistasis; Goals; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Heterotrimeric GTP-Binding Proteins; Human; Hydrolysis; IRAK1 gene; Impairment; In Vitro; Intervention; Lead; Ligand Binding; Ligands; Malignant Neoplasms; Mediating; Modernization; Molecular; Mouse-ear Cress; Mutation; Nature; Opioid Analgesics; Pathway interactions; Pharmaceutical Preparations; Phenocopy; Phenotype; Phosphorylation; Phosphotransferases; Plant Model; Plant Proteins; Plants; Post-Translational Protein Processing; Process; Protein Conformation; Protein Subunits; Proteins; Proteomics; RGS Proteins; RGS1 gene; Recombinants; Regulation; Research; Seedling; Signal Transduction; Signaling Protein; Site; Specificity; Stress; System; Testing; Transmembrane Domain; Variant; Whole Organism; base; dimer; drug discovery; environmental stressor; experimental study; genetic analysis; genetic resource; hormonal signals; improved; in vitro Assay; in vivo; member; mutant; novel; organ growth; pathogen; phosphoproteomics; protein function; protein protein interaction; receptor; repaired; response; small molecule; tool

Project Summary Heterotrimeric guanine nucleotide-binding (G) proteins composed of Gα, Gβ, and Gγ subunits function as molecular switches in signal transduction. Human G protein pathways are targets of over 30% of clinical drugs and their mutation causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. While humans have 16 Gα, 5 Gβ, and 12 Gγ genes, the model plant, Arabidopsis thaliana, has many fewer -- only one canonical Gα (GPA1), one Gβ (AGB1), and three Gγ (AGG1, AGG2 and AGG3) genes. Yet plant G proteins, as in humans, regulate a multiplicity of pathways. Arabidopsis G protein mutants show developmental defects, defects in pathogen defense, and defects in responses to environmental stresses. While Arabidopsis has only one protein (GCR1) with sequence and structural similarity to the 800+ metazoan GPCRs, Arabidopsis has ~600 receptor-like-kinases (RLKs), which form a monophyletic group with Pelle kinases of metazoans. Mass spectrometric analyses in the PI's lab have revealed multiple RLKs present in GPA1 and AGB1 immunoprecipitates, consistent with new genetic evidence of RLK/G protein interaction and confirmed by our kinase assays showing direct GPA1 phosphorylation by RLKs. Phosphoproteomics identifies G protein subunit phosphorylation at conserved sites in plant and metazoan G protein subunits. The overarching hypothesis of the proposed research is that phosphorylation of G protein subunits is an evolutionarily conserved regulatory mechanism that confers specificity to G protein signaling. Three explicit hypotheses are: 1) phosphorylation of G protein subunits controls G protein guanine nucleotide exchange, GTPase activity and heterotrimer association; 2) ligand-activated kinases (RLKs in plants) are GPCRs at the apex of phospho-controlled G protein signaling networks, and; 3) Gα and Gβγ subunit phosphorylation confers specificity to G protein subunit interaction with downstream partners (effectors). These hypotheses will be tested in the Arabidopsis system by, respectively: 1) biochemical assays of the G protein cycle and protein- protein interactions within the heterotrimer; 2) genetic epistasis analysis between G protein subunits and implicated RLKs, phosphoproteomic analyses of G protein subunits following RLK ligand stimulation and in rlk null lines, and in vitro phosphorylation assays; 3) phenotyping of G protein subunit null mutants complemented with phosphomimic and phosphonull G protein subunits, identification of phosphorylation-dependent subunit- effector interactions by coimmunoprecipitation and protein-protein interaction assays, and targeted tests of effector competition for G protein subunits. Arabidopsis is ideal to address these issues in a systematic and comprehensive manner, as it provides a limited number of G protein subunits, ease of manipulation, a plethora of genetic tools, and an array of whole-organism and single-cell G protein phenotypes. Completion of these goals will reveal new mechanisms of G protein modulation, amenable for manipulation and drug targeting to meet the major challenge of pathway-specific control of G protein signaling for improvement of human health.

项目摘要 由Gα、Gβ和Gγ亚基组成的异源三聚体鸟嘌呤核苷酸结合（G）蛋白的功能是 信号转导中的分子开关。人类G蛋白通路是超过30%的临床药物的靶点 它们的突变导致遗传疾病、发育异常和改变的传染病 易感性虽然人类有16个Gα，5个Gβ和12个Gγ基因，但模式植物拟南芥有 只有一个典型的Gα（GPA 1），一个Gβ（AGB 1）和三个Gγ（AGG 1，AGG 2和AGG 3）基因。 然而，植物G蛋白和人类一样，调节着多种途径。拟南芥G蛋白突变体显示 发育缺陷、病原体防御缺陷和对环境应激反应的缺陷。 而拟南芥只有一个蛋白质（GCR 1）与800+后生动物的序列和结构相似， 拟南芥有大约600种受体样激酶（RLKs），与Pelle形成单系群 后生动物的激酶。PI实验室的质谱分析显示， GPA 1和AGB 1免疫沉淀，与RLK/G蛋白相互作用的新遗传证据一致， 通过我们的激酶测定证实，显示RLK直接磷酸化GPA 1。磷酸化蛋白质组学鉴定 植物和后生动物G蛋白亚基保守位点的磷酸化。的 这项研究的首要假设是，G蛋白亚基的磷酸化是一种 进化上保守的调节机制，赋予G蛋白信号传导特异性。三明确 假设是：1）G蛋白亚基的磷酸化控制G蛋白鸟嘌呤核苷酸交换， 配体激活激酶（植物中的RLK）是GPCR，其位于GPCR的下游。 磷酸化控制的G蛋白信号网络的顶点，和3）Gα和Gβγ亚基磷酸化赋予 G蛋白亚基与下游伴侣（效应子）相互作用的特异性。这些假设将是 在拟南芥系统中分别通过以下方法进行测试：1）G蛋白循环和蛋白质- 异源三聚体内的蛋白质相互作用; 2）G蛋白亚基和 涉及RLK，在RLK配体刺激后G蛋白亚基的磷酸化蛋白质组学分析和rlk中 空株和体外磷酸化测定; 3）G蛋白亚基空突变体的表型分析， 磷酸化依赖性亚基的鉴定， 通过免疫共沉淀和蛋白质-蛋白质相互作用测定的效应物相互作用，以及 G蛋白亚基的效应子竞争。拟南芥是解决这些问题的理想系统， 全面的方式，因为它提供了有限数量的G蛋白亚基，易于操作，过多的 基因工具，以及一系列的全生物体和单细胞G蛋白表型。完成这些 这些目标将揭示G蛋白调节的新机制，可用于操纵和药物靶向， 满足G蛋白信号传导的途径特异性控制以改善人类健康的主要挑战。