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: 8908573
• 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/8908573
• 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; 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; public health relevance; 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(申请人提供)：G蛋白偶联受体(GPCRs)在不同的信号通路中发挥关键作用，具有重要的生理学和医学意义。GPCR介导的信号转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，导致亚单位解离和下游效应器的激活。然而，不同配体结合导致特定信号输出的潜在生化机制仍然知之甚少。酵母信息素反应途径作为研究GPCR信号系统一般性质的遗传易操作和表征良好的模型的研究揭示了受体行为的一些意想不到的方面，这些方面与将信号输出与配体结合输入联系起来的简单模型不一致。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，考虑到疗效和效力，不受受体表达在~100倍受体水平上的变化的影响；2)观察到，特征良好的信息素反应途径的拮抗剂，当与激动剂混合时，在很大程度上未能抑制信号，抑制程度将根据激动剂和受体拮抗剂的已知亲和力预测；3)观察到，拮抗剂与Ste2p受体结合可刺激受体内化，尽管它们不会刺激G蛋白介导的反应。综上所述，这些发现支持这样一种观点，即受体的行为比简单的开和关状态之间的平衡复杂得多，尤其是GPCR信号中存在一个重要的负面因素，即在缺乏配体的情况下，抑制G蛋白的激活，但这种情况可能被拮抗剂与受体结合而逆转。为了理解这些发现，建议的方法包括测试失活受体和G蛋白之间的物理联系，鉴定特定改变所述行为的突变途径组件，以及表征和操纵拮抗剂依赖的内吞作用的机制。我们还将通过对寡聚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特性，来研究受体-受体相互作用的功能作用。综上所述，这些结果有望提供一个更现实和更详细的视角，将信号响应与受体的配体占位联系起来，有望适用于其他GPCR途径。