Mechanisms of G Protein Coupled Receptor Signaling in the Yeast Pheromone Pathway

酵母信息素途径中 G 蛋白偶联受体信号传导机制

基本信息

批准号：
9045646
负责人：
MARK E. DUMONT
金额：
$ 29.17万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9045646
关键词：
Affect Affinity Agonist Beds Behavior Binding Binding Sites Biochemical Biological Models Cell Surface Receptors Cell surface Cells Complex Coupling Defect Dependence Detection Dissociation Elements Endocytosis Energy Transfer Equilibrium Event Family Fluorescence G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Genetic Genetic Screening Health Heterotrimeric GTP-Binding Proteins Homo Hormonal Knowledge Label Ligand Binding Ligands Mediating Medicine Modeling Mutate Nucleotides Output Partner in relationship Pathway interactions Pharmaceutical Preparations Pheromone Pheromone Receptors Physiology Play Population Process Property Proteins Receptor Signaling Regulation Reporting Role Sensory Signal Pathway Signal Transduction Stimulus Study models Surface System Testing Variant Yeasts base biochemical tools design expectation member mutant protein activation receptor receptor binding receptor expression receptor internalization receptor-mediated signaling response trafficking

项目摘要

DESCRIPTION (provided by applicant): G Protein Coupled Receptors (GPCRs) play critical roles in diverse signaling pathways with important implications for physiology and medicine. The basic outlines of GPCR-mediated signaling are well known- agonist binding to receptors stimulates nucleotide exchange on a trimeric G protein, leading to subunit dissociation and activation of downstream effectors. However, aspects of the underlying biochemical mechanisms by which binding of different ligands leads to particular signaling outputs remains poorly understood. Study of the yeast pheromone response pathway as a genetically tractable and well-characterized model for studying general properties of GPCR signaling systems have revealed unexpected aspects of the receptor behavior that are not consistent with simple models relating signaling output to ligand-binding input. These include: 1) the observation that signaling responses to binding of agonist to the pheromone receptor Ste2p, considering both efficacy and potency, are not affected by changes in receptor expression over an ~100 fold range of receptor levels; 2) the observation that well characterized antagonists of the pheromone response pathway, when mixed with agonist, fail by a large margin to inhibit signaling to the extent that would be predicted based on known affinities of the agonist and antagonist for receptors; 3) the observation that binding of antagonists to Ste2p receptors stimulates receptor internalization even though they do not elicit stimulation of G protein mediated responses. Taken together, these findings support the idea that receptor behavior is much more complex than a simple equilibrium between on and off states and, in particular, that there is a significant negative element to GPCR signaling that, in the absence of ligand, inhibits G protein activation, but that may be reversed by binding of antagonist to receptors. The proposed approaches for understanding these findings include testing for physical association between inactivated receptors and G proteins, identification of mutated pathway components that specifically alter the described behaviors, and characterization and manipulation of the mechanisms of the antagonist-dependent endocytosis. We will also investigate the functional role of receptor-receptor interactions by conducting a fluorescence-based genetic screen for receptors with specific defects in oligomerization and analyzing the signaling properties of the mutant receptors. Taken together, the results are expected to provide a more realistic and detailed view of the processes that relate signaling responses to ligand occupancy of receptors that can be expected to be applicable to other GPCR pathways.

描述（由适用提供）：G蛋白偶联受体（GPCR）在潜水员信号通路中起关键作用，对生理和医学具有重要意义。 GPCR介导的信号传导的基本轮廓是众所周知的与受体的结合，刺激了三聚体G蛋白上的核丁基交换，从而导致亚基解离并激活下游效应。但是，通过不同配体的结合导致特定信号输出的基本生化机制的各个方面仍然知之甚少。研究酵母信息素响应途径是研究GPCR信号系统的一般特性的一般易处理和良好的模型，这表明受体行为的意外方面与将信号输出与配体结合输入相关的简单模型不一致。其中包括：1）观察到，考虑到有效性和效力，激动剂与信息素受体Ste2p结合的信号传导响应不受受体表达的变化的影响。 2）观察到，当与激动剂混合时，良好表征的源代酮反应途径的拮抗剂会大大限制，无法抑制信号传导，以基于受体的激动剂和拮抗剂的已知亲和力预测的程度。 3）观察到拮抗剂与Ste2p受体的结合刺激受体内在化，即使它们不引起G蛋白介导的反应的刺激。综上所述，这些发现支持了以下观点：受体行为比在远离状态和关闭状态之间的简单等效术更复杂，尤其是，GPCR信号存在显着的负元素，即在没有配体的情况下，抑制G蛋白的激活，但可以通过拮抗剂与受体的结合来逆转。理解这些发现的建议方法包括测试失活的受体和G蛋白之间的物理关联，对特异性改变所描述行为的突变途径成分的鉴定，以及对拮抗剂依赖性内吞作用机制的表征和操纵。我们还将通过对基于荧光的受体进行基于荧光的受体进行寡聚化和分析突变受体的信号传导特性来研究受体受体相互作用的功能作用。综上所述，结果有望提供对将信号响应与受体配体占用率相关的过程的更现实，更详细的视图，这些响应可预期适用于其他GPCR途径。