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

 描述（由申请人提供）：G蛋白偶联受体（GPCR）在多种信号通路中发挥关键作用，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-激动剂与受体的结合刺激三聚体G蛋白上的核苷酸交换，导致亚基解离和下游效应物的激活。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方面仍然知之甚少。研究酵母信息素反应途径作为一种遗传上易于处理和良好表征的模型，用于研究GPCR信号系统的一般性质，揭示了受体行为的意想不到的方面，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。其中包括：1）观察到对激动剂与信息素受体Ste 2 p结合的信号传导响应，考虑到功效和效力，不受受体表达在受体水平的~100倍范围内的变化的影响; 2）观察到，当与激动剂混合时，在很大程度上不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Ste 2 p受体的结合刺激受体内化，即使它们不引起G蛋白介导的应答的刺激。总之，这些发现支持了这样的观点，即受体行为比开和关状态之间的简单平衡要复杂得多，特别是，存在对GPCR信号传导的显著负元件，其在不存在配体的情况下抑制G蛋白活化，但可通过拮抗剂与受体的结合而逆转。理解这些发现的建议方法包括测试失活受体和G蛋白之间的物理关联，鉴定特异性改变所述行为的突变途径组分，以及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。两者合计，结果预计将提供一个更现实的和详细的视图的过程中，相关的信号响应，配体占用的受体，可以预期适用于其他GPCR途径。

 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.