Direct assessment of GPCR-transducer coupling and G protein subtype bias

项目摘要/摘要 G蛋白偶联受体(GPCRs)是激素、神经递质和大约 FDA批准的三分之一的药物。这些受体通过与四个家族的转导蛋白偶联来传递信号。 异源三聚体G蛋白和四个阻滞剂家族。现在人们认识到，个别的GPCR产生 通过与几个不同的换能器亚型相互作用来进行复杂的信号响应，即信号是 “多维”。给定的GPCR对不同转导蛋白的广泛选择性决定了它的能力 影响细胞行为，因此知道哪些转导能被每个转导激活是重要的 受体。这种信息可能很难获得，这在很大程度上是因为换能器耦合 通常使用监测受体下游的事件的分析来间接推断，如 作为第二信使的积累。还可以理解，某些激活配体(激动剂)可以 特别是以牺牲其他换能器为代价，促进与一些换能器的GPCR耦合，这是最常见的现象 被称为“有偏见的激励主义”。有偏向的激动剂特别有希望成为药物开发的候选药物， 并已迅速进入临床环境，因为它们能够产生治疗反应 而不会产生不利的影响。到目前为止，几乎所有发现的有偏见的激动剂都在G 蛋白质和阻滞素是一类，很少有配体被认为是在不同的G 蛋白质亚型。再一次，发现和发展G蛋白亚型选择性的关键障碍 药物依赖于下游信号分析，而下游信号分析缺乏必要的灵敏度来检测细微之处。 配基偏置，易受信号串扰影响。我们将使用以下组合来解决这些问题 先进的光学工具，允许直接评估接收器-换能器耦合。具体地说，我们将使用 改进了基于工程迷你G(Mg)蛋白的细胞和无细胞分析：1)全面 用于&gt；200非感应性GPCR的GPCR型换能器联轴器；2)表征和发现新的G 蛋白质亚型-选择性配体。这些研究将产生和测试对以下方面至关重要的假设 多维信号，将使一套强大的新的敏感换能器分析可用于 科学界，并将促进对G蛋白亚型偏向配体的潜在理解 治疗剂。

PROJECT SUMMARY/ABSTRACT G protein-coupled receptors (GPCRs) are important targets of hormones, neurotransmitters and approximately one-third of FDA-approved drugs. These receptors signal by coupling to transducer proteins from four families of heterotrimeric G proteins and a family of four arrestins. It is now recognized that individual GPCRs produce complex signaling responses by interacting with several different transducer subtypes, i.e. that signaling is “pluridimensional”. The broad selectivity of a given GPCR for different transducer proteins defines it's ability to influence cellular behavior, therefore it is important to know which tranducers can be activated by each receptor. This information can be surprisingly difficult to obtain, in large part because transducer coupling is most often inferred indirectly using assays that monitor events that are well downstream from receptors, such as accumulation of second messengers. It is also understood that certain activating ligands (agonists) can specifically promote GPCR coupling to some transducers at the expense of others, a phenomenon most often referred to as “biased agonism”. Biased agonists are especially promising candidates for drug development, and have rapidly advanced into the clinical setting, because they are able to produce therapeutic responses without also producing adverse effects. Almost all biased agonists discovered to date select between G proteins and arrestins as classes, and there are very few ligands that are thought to select between different G protein subtypes. Again, a critical roadblock to discovery and development of G protein subtype-selective drugs is the reliance on downstream signaling assays, which lack the necessary sensitivity to detect subtle ligand bias and are susceptible to signal crosstalk. We will address these problems using a combination of advanced optical tools that allow direct assessment of receptor-transducer coupling. Specifically, we will use improved cellular and cell-free assays based on engineered mini G (mG) proteins to: 1) comprehensively profile GPCR transducer coupling for >200 non-sensory GPCRs, and 2) characterize and discover new G protein subtype-selective ligands. These studies will generate and test hypotheses that are central to pluridimensional signaling, will make a powerful new set of sensitive transducer assays available to the scientific community, and will advance understanding of G protein subtype-biased ligands as potential therapeutic agents.