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

• 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
• 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

 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（由申请人提供）：G 蛋白偶联受体 (GPCR) 在多种信号传导途径中发挥着关键作用，对生理学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——激动剂与受体结合刺激三聚体 G 蛋白上的核苷酸交换，导致亚基解离和下游效应器的激活。然而，不同配体的结合导致特定信号输出的潜在生化机制的各个方面仍然知之甚少。将酵母信息素响应途径作为研究 GPCR 信号系统一般特性的遗传易处理且特征良好的模型进行研究，揭示了受体行为的意外方面，这些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。这些包括：1) 观察到激动剂与信息素受体 Ste2p 结合的信号反应，考虑到功效和效力，不受受体水平约 100 倍范围内受体表达变化的影响； 2) 观察到，当与激动剂混合时，信息素响应途径的充分表征的拮抗剂在很大程度上未能抑制信号传导至基于激动剂和拮抗剂对受体的已知亲和力所预测的程度； 3)观察到拮抗剂与Ste2p受体的结合刺激受体内化，即使它们不引起G蛋白介导的反应的刺激。总而言之，这些发现支持这样的观点，即受体行为比开关状态之间的简单平衡要复杂得多，特别是，GPCR 信号传导存在显着的负面因素，在没有配体的情况下，会抑制 G 蛋白激活，但可以通过拮抗剂与受体的结合来逆转这种情况。所提出的理解这些发现的方法包括测试失活受体和 G 蛋白之间的物理关联，识别特异性改变所描述行为的突变途径成分，以及拮抗剂依赖性内吞作用机制的表征和操作。我们还将通过对寡聚化中具有特定缺陷的受体进行基于荧光的遗传筛选并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。总而言之，这些结果预计将为将信号传导反应与受体的配体占据相关的过程提供更现实和更详细的视图，预计这些过程也适用于其他 GPCR 途径。