The quiesome and signalosome of a model GPCR

GPCR 模型的安静和信号体

基本信息

批准号：
8787890
负责人：
JEFFREY M. BECKER
金额：
$ 27.63万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8787890
关键词：
Accounting Affinity Amino Acids Biological C-terminal Cell Extracts Cell membrane Cell surface Cells Complement Coupled Cytoplasmic Tail Diabetes Mellitus Digestion Disease Drug Design Drug Prescriptions Engineering Event Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Heterotrimeric GTP-Binding Proteins Hormones Human Genome Immunoprecipitation Integral Membrane Protein Knowledge Lasers Life Ligand Binding Ligands Light Malignant Neoplasms Mass Spectrum Analysis Mediating Mediation Medicine Methods Modeling Partner in relationship Pathology Peptides Pharmaceutical Preparations Pharmacotherapy Pheromone Receptors Positioning Attribute Protein Family Proteins Receptor Activation Receptor Mediated Signal Transduction Recruitment Activity Regulation Rest Saccharomyces cerevisiae Scanning Series Signal Pathway Signal Transduction Signaling Molecule Stimulus Structure System Techniques Time Transmembrane Domain Yeasts analog base benzoylphenylalanine crosslink design effective therapy innovation mutant novel protein complex protein protein interaction public health relevance receptor receptor function receptor structure function

Accounting Affinity Amino Acids Biological C-terminal Cell Extracts Cell membrane Cell surface Cells Complement Coupled Cytoplasmic Tail Diabetes Mellitus Digestion Disease Drug Design Drug Prescriptions Engineering Event Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Heterotrimeric GTP-Binding Proteins Hormones Human Genome Immunoprecipitation Integral Membrane Protein Knowledge Lasers Life Ligand Binding Ligands Light Malignant Neoplasms Mass Spectrum Analysis Mediating Mediation Medicine Methods Modeling Partner in relationship Pathology Peptides Pharmaceutical Preparations Pharmacotherapy Pheromone Receptors Positioning Attribute Protein Family Proteins Receptor Activation Receptor Mediated Signal Transduction Recruitment Activity Regulation Rest Saccharomyces cerevisiae Scanning Series Signal Pathway Signal Transduction Signaling Molecule Stimulus Structure System Techniques Time Transmembrane Domain Yeasts analog base benzoylphenylalanine crosslink design effective therapy innovation mutant novel protein complex protein protein interaction public health relevance receptor receptor function receptor structure function

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项目摘要

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the largest integral membrane protein family in the human genome. These receptors function to transduce signals across the plasma membrane allowing cells to respond and adapt to a variety of different stimuli, such as light, odorants, hormones and many other signaling molecules. This diverse family of receptors is intimately associated with a variety of disease states, including cancer and diabetes, making them attractive targets for drug therapy; it is estimated that 30-50% of prescribed medications target pharmaceutically relevant GPCRs. The aim of this proposal is to identify intracellular proteins that regulate and modulate Ste2p, a yeast receptor that has been used widely as a model for GPCR structure and function. We propose to bridge a large gap in our knowledge of the initial events of GPCR-mediated signal transduction by identifying the "quiesome" (proteins associated with the receptor in the quiescent or resting state) and the "signalosome" (intracellular proteins which are recruited and interact with GPCRs upon activation). To accomplish these aims, we will utilize unnatural amino acid replacement to genetically modify Ste2p to contain benzoylphenylalanine (Bpa), a photoactivatable, unnatural amino acid to allow the capture of proteins associated with the intracellular domains of Ste2p in intact, living cells in the presence and absence of its peptide ligand. Our hypothesis is that specific proteins interact with intracellular domains of Ste2p, that these proteins can be captured by the use of laser activation of Bpa in order to generate a time-resolved picture of the intracellular protein partners which will be identified by mass spectrometry, and that our methods using live cells, laser activation will yield a more accurate and dynamic account of GPCR protein partners compared to other methods used to date such as immunoprecipitation. It is expected that the experimental techniques proposed herein will be applicable to medically important GPCRs and that the description of the quiesome and signalosome will provide new targets for drug design leading to new and effective therapies for diseases involving GPCRs.

描述（由申请人提供）：G蛋白偶联受体（GPCR）是人基因组中最大的整体膜蛋白家族。这些受体的功能在跨质膜上传导信号，从而使细胞能够反应并适应各种不同的刺激，例如光，气味，激素和许多其他信号分子。这种多样化的受体系列与包括癌症和糖尿病在内的各种疾病状态密切相关，使它们成为药物治疗的吸引力。据估计，有30％至50％的处方药针对药物相关的GPCR。该建议的目的是鉴定调节和调节Ste2p的细胞内蛋白质，这是一种已广泛用作GPCR结构和功能模型的酵母受体。我们建议通过识别“ quiesome”（与静止状态或静止状态的受体相关的蛋白质）和“信号体”（激活后募集并与GPCR相互作用），建议通过识别“ quiesome”（与受体相关的蛋白质）来了解GPCR介导的信号转导的初始事件的大差距。为了实现这些目标，我们将利用不自然的氨基酸替代替代基因修饰Ste2p来含有苯甲酰甲基丙氨酸（BPA），这是一种可光活化的，不自然的氨基酸，允许捕获蛋白质与Ste2p在Intact中的细胞内的细胞内的细胞相关的蛋白质，并在其存在和吸收lig lig lig lig lig lig lig lig lig lig。我们的假设是特定的蛋白质与Ste2p的细胞内结构域相互作用，可以捕获这些蛋白通过使用激光激活BPA，以生成细胞内蛋白质伴侣的时间分辨图片，将通过质谱法鉴定，并且我们使用活细胞的方法，激光激活将产生与其他与其他方法相比的GPCR蛋白伴侣的更准确和动态的说明，而不是使用该方法。预计本文提出的实验技术将适用于医学上重要的GPCR，并且对Quiesome和Signalsosom的描述将为药物设计提供新的靶标，从而为涉及GPCR的疾病提供新的有效疗法。