Trimeric G proteins as Novel Targets in Cancer Progression

三聚体 G 蛋白作为癌症进展的新靶点

• 批准号: 9259100
• 负责人: Nicholas Antonios Kalogriopoulos
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9259100
• 关键词: Affect; American; Anatomy; Binding; Biochemical; Biochemistry; Biological Assay; Biological Process; Breast Cancer Cell; Cells; Code; Complex; Computer Simulation; Couples; Disease; Enzymatic Biochemistry; Fluorescence Resonance Energy Transfer; Functional disorder; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Genetic; Goals; Growth; Growth Factor; Growth Factor Receptors; Guanidines; Guanine Nucleotide Dissociation Inhibitors; Guanine Nucleotide Exchange Factors; Health; Heterotrimeric GTP-Binding Proteins; Histidine; Human; Image; In Vitro; Intercept; Ions; Lead; Ligands; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Membrane; Mentors; Mitosis; Molecular; Monomeric GTP-Binding Proteins; Mutate; Mutation; Neoplasm Metastasis; Pathway interactions; Pattern; Peptides; Permeability; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Phosphotyrosine; Property; Protein Chemistry; Protein Tyrosine Kinase; Proteins; Publishing; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recruitment Activity; Reporting; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Somatic Mutation; Structure; Technology; Therapeutic; Therapeutic Intervention; Transactivation; Transducers; Treatment Efficacy; Tumor Cell Invasion; Tyrosine; Tyrosine Phosphorylation; Vesicle; Work; X-Ray Crystallography; cancer cell; cancer therapy; cell behavior; cell motility; design; experimental study; in vivo; insight; migration; molecular targeted therapies; mutant; neoplastic cell; novel; protein activation; protein protein interaction; receptor; therapeutic target; tumor; tumor progression; tumorigenesis

ABSTRACT: Heterotrimeric G proteins are molecular switches that control signal transduction. Dysregulation of the G protein pathway can lead to aberrant signal transduction and herald many diseases and oncogenesis. Although G proteins are traditionally known to transduce signals initiated by G protein coupled receptors (GPCRs), a growing body of work by my mentor's group and others have established that they also transduce signaling downstream of yet another large group of receptors, the growth factor receptor tyrosine kinases (RTKs), via mechanisms that are poorly understood. Recent studies have demonstrated that Gα-Interacting Vesicle associated protein (GIV, a.k.a Girdin) is an unusual signal transducer that can bind both RTKs and G proteins. As a direct consequence of an unusual modular makeup of GIV, signals initiated by multiple RTKs converge on the GIV-platform to trigger non-canonical transactivation of trimeric G protein, Gαi. Working downstream of a variety of growth factors and ligands, it has been demonstrated that the consequences of such signaling are far reaching, and that the impact on a diverse set of biological processes, in both health and disease, is enormous. Despite the insights gained, the mechanism of G protein activation in close proximity of RTKs remains unclear, how may this pathway affect signal transduction or cellular phenotype, and what might be the structural basis for this unusual RTK-GIV-Gi pathway and their pathophysiologic consequences remain unexplored. Preliminary results indicate that the proximity between the receptor and the G protein is essential for phosphorylation of Gαi by multiple RTKs at three unique tyrosines, that such phosphorylation requires GIV to recruit G proteins to the RTKs, and that one of the major. These findings will be studied in-depth through the experiments in the following 3 aims – 1) assess the consequence(s) of phosphorylation of Gαi by multiple growth factor RTKs using in vitro and in vivo phosphorylation assays, protein-protein interaction assays with various modulators of G proteins, measures of Gi activation, and phenotypic assays to evaluate migration, invasion, mitosis, and survival in cells expressing WT or Y mutants of Gi; 2) investigate how transactivation of G proteins by RTKs is deregulated in cancers by studying the profile of RTK-triggered tyrosine phosphorylation of Gαi in tumor cells during metastasis and by characterization of a novel somatic mutation in Gαi where the tyr (Y) that is targeted by RTKs is mutated to his (H) using similar biochemical and cell biological assays as outlined in Aim 1; and 3) elucidate the structural basis for phosphotyrosine-dependent transactivation of G proteins using a combination of protein chemistry, functional binding assays with rationally designed mutant proteins, and x-ray crystallography. The overall goal of this proposal is to dissect the mechanisms by which multiple growth factor RTKs transactivate trimeric G proteins via the novel linker/platform, GIV from an atomic level to tumor cell phenotype.

抽象的： 异三聚体 G 蛋白是控制信号转导的分子开关。 G失调 蛋白质途径可导致异常信号转导并预示许多疾病和肿瘤发生。 尽管传统上已知 G 蛋白可转导由 G 蛋白偶联受体引发的信号 （GPCR），我的导师小组和其他人所做的越来越多的工作已经证实它们也可以转换 另一大类受体（生长因子受体酪氨酸激酶）下游的信号传导 （RTK），通过人们知之甚少的机制。最近的研究表明，Gα-相互作用 囊泡相关蛋白（GIV，又名 Girdin）是一种不寻常的信号转导器，可以结合 RTK 和 G 蛋白质。作为 GIV 不寻常的模块化构成的直接结果，由多个 RTK 发起的信号 汇聚在 GIV 平台上，触发三聚体 G 蛋白 Gαi 的非规范反式激活。在职的 在多种生长因子和配体的下游，已经证明， 这种信号影响深远，对健康和健康等多种生物过程产生影响 疾病，是巨大的。尽管获得了一些见解，但 G 蛋白激活的机制与 RTK 仍不清楚，该途径如何影响信号转导或细胞表型，以及什么可能 是这种不寻常的 RTK-GIV-Gi 途径的结构基础，其病理生理学后果仍然存在 未经探索。初步结果表明受体和 G 蛋白之间的接近性至关重要 对于通过多个 RTK 在三个独特的酪氨酸处磷酸化 Gαi，这种磷酸化需要 GIV 将 G 蛋白招募到 RTK，这是​​主要之一。这些发现将通过以下方式进行深入研究： 以下 3 个目标的实验 – 1) 通过多重评估 Gαi 磷酸化的后果 使用体外和体内磷酸化测定、蛋白质-蛋白质相互作用测定的生长因子 RTK G 蛋白的各种调节剂、Gi 激活的测量以及评估迁移的表型测定， 表达Gi WT或Y突变体的细胞的侵袭、有丝分裂和存活； 2）研究反式激活如何 通过研究 RTK 触发的酪氨酸谱，RTK 对 G 蛋白的调节在癌症中失调 肿瘤细胞转移过程中 Gαi 的磷酸化以及新体细胞突变的表征 Gαi，其中 RTK 靶向的 tyr (Y) 使用类似的生化和细胞生物学突变为 his (H) 目标 1 中概述的测定； 3）阐明磷酸酪氨酸依赖性的结构基础 结合蛋白质化学、功能结合测定法对 G 蛋白进行反式激活 合理设计的突变蛋白和 X 射线晶体学。该提案的总体目标是剖析 多种生长因子 RTK 通过新型反式激活三聚体 G 蛋白的机制 连接子/平台，GIV从原子水平到肿瘤细胞表型。