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（申请人证明）：G蛋白耦合受体（GPCR）在重要的SINGIAL和医学中发挥着关键作用。下游效应子的亚基分解。简单的模型将输出与配体结合输入有关。一个很大的边缘抑制了基于激动剂预测的已知激动剂的程度，即使拮抗剂与ste2p接收器结合，即使它们没有引起甲状腺素D的响应，这些受体比简单的复杂性更为复杂。尤其是，均衡是GPCR信号的显着负元素，在没有配体的情况下，固有的G蛋白激活，拮抗剂与受体的结合。蛋白质的行为以及对拮抗剂依赖性内吞作用的表征和操纵。预计结果将提供并详细的视图，这些过程将信号响应与配体占用OR相关联的过程，这些响应可预期适用于其他GPCR途径。