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.

摘要： 异源三聚体G蛋白是控制信号转导的分子开关。G调节失调 蛋白质通路可导致异常的信号转导，并预示着许多疾病和肿瘤的发生。 虽然传统上已知G蛋白阻断由G蛋白偶联受体启动的信号 （GPCRs），我的导师的团队和其他人越来越多的工作已经确定，他们也 在另一大类受体的下游信号传导，生长因子受体酪氨酸激酶 （RTK），通过机制知之甚少。最近的研究表明，Gα相互作用 囊泡相关蛋白（GIV，a.k.a. Girdin）是一种不寻常的信号转导蛋白，它可以结合RTK和G proteins.作为GIV不寻常的模块化构成的直接结果， 聚集在GIV平台上以触发三聚体G蛋白Gαi的非经典反式激活。工作 在多种生长因子和配体的下游，已经证明， 这种信号是深远的，并且对各种生物过程的影响，在健康和 疾病，是巨大的。尽管获得了这些见解，但G蛋白在极近距离激活的机制 RTKs仍然不清楚，这条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通路的结构基础，其病理生理后果仍然存在 未开发的初步结果表明，受体和G蛋白之间的接近是必要的 对于Gαi在三个独特的酪氨酸上被多个RTK磷酸化，这种磷酸化需要GIV 为RTK募集G蛋白，这是一个主要的。这些研究结果将通过 以下3个目的的实验- 1）评估Gαi的磷酸化的后果， 生长因子RTK使用体外和体内磷酸化测定，蛋白质-蛋白质相互作用测定， G蛋白的各种调节剂，Gi活化的测量，和评估迁移的表型测定， 在表达Gi的WT或Y突变体的细胞中的侵袭、有丝分裂和存活; 2）研究反式激活如何在细胞中表达。 通过研究RTK触发的酪氨酸蛋白酶谱， 转移过程中肿瘤细胞中Gαi磷酸化，并通过表征肿瘤细胞中一种新的体细胞突变， Gαi，其中使用类似的生物化学和细胞生物学方法将RTK靶向的tyr（Y）突变为his（H） 目的1中概述的测定;和3）阐明磷酸酪氨酸依赖性 G蛋白的反式激活，使用蛋白质化学、功能性结合测定与 合理设计的突变蛋白和X射线晶体学。这项提案的总体目标是剖析 多种生长因子RTKs通过新的转录激活三聚体G蛋白的机制， 接头/平台，从原子水平到肿瘤细胞表型的GIV。