Probing molecular mechanisms of GPCR functional selectivity in live cells

描述（由申请人提供）：虽然最近的几项研究报告了不同的配体依赖性GPCR构象，但仍不清楚这些构象如何引起差异下游响应。相比之下，从功能反应中解读G蛋白选择是复杂的，因为调节GPCR信号传导的无数因素，包括GPCR、G蛋白的相对丰度和可用性，定位于不同的膜表面或微结构域，以及调节蛋白如支架、激酶、抑制蛋白和细胞内吞装置的影响。拟议的研究通过PI开发的一类新的基于FRET的SPASM传感器来解决这些限制，以系统地调节活细胞中的蛋白质-蛋白质相互作用。研究将集中在四种GPCR，β 2-肾上腺素受体（β 2-AR），α 2A-肾上腺素受体，黑皮质素4受体和β 3-肾上腺素受体，其差异G蛋白选择在心力衰竭，肥胖和糖尿病的进展和治疗中具有重要的功能后果。 目的1中的研究利用检测GPCR与源自Ga亚基c-末端的肽（GPCR-G蛋白配对的已知决定子）之间的相互作用的传感器。基于FRET的测量将与已建立的方法相结合，以测试GPCR以配体依赖性方式采用功能上不同的构象以触发差异下游响应的假设。在初步研究中，用β 2-AR测试传感器测量的特异性，并导致鉴定由β受体阻滞剂美托洛尔触发的功能性Gi构象。研究的结构，剖析不同的事件，GPCR-G蛋白相互作用，然后刺激与规范和偏见激动剂。 从FRET/BRET研究中对GPCR-G蛋白相互作用和G蛋白选择的清楚理解受到缺乏相对浓度控制的限制。目的2中的研究将利用SPASM技术来描述配体刺激前后的事件顺序。解决的关键问题是：（1）无配体的GPCR是否与G蛋白基本相关或预偶联？(2)配体刺激后GPCR是否从G蛋白中解离？(3)G蛋白的选择是否与GPCR对G蛋白的固有结合亲和力相关？(4)GPCR和G蛋白的局部浓度在多大程度上影响偏性信号传导？使用SPASM技术的GPCR-G蛋白相互作用浓度的系统变化将用于评估FRET和下游响应的特异性。 拟议的研究有可能为未来研究任何GPCR引发的配体驱动的信号传导创造一个新的技术和概念平台。SPASM传感器的广泛验证是本研究的一个组成部分，这反过来将为它们在活细胞药物筛选中的应用铺平道路，以识别使GPCR信号偏向治疗所需结果的小分子。

DESCRIPTION (provided by applicant): While several recent studies have reported on distinct ligand-dependent GPCR conformations, it remains unclear how these conformations elicit differential downstream responses. In contrast, deciphering G protein selection from functional response is complicated by the myriad of factors that regulate GPCR signaling, including relative abundance and availability of GPCR, G proteins, localization to different membrane surfaces or micro-domains, and the influence of regulatory proteins such as scaffolds, kinases, arrestins and the cellular endocytic apparatus. The proposed research addresses these limitations with a novel class of FRET-based SPASM sensors developed by the PI to systematically modulate protein-protein interactions in live cells. Research will focus o four GPCRs, b2-adrenoceptor (b2-AR), a2A-adrenoceptor, melanocortin4 receptor and b3-adrenoceptor, whose differential G protein selection has important functional consequences in the progression and treatment of heart failure, obesity and diabetes. Studies in aim 1 utilize sensors that detect the interaction between a GPCR and a peptide derived from the Ga subunit c-terminus, a known determinant of GPCR-G protein pairing. FRET-based measurements will be combined with established approaches to test the hypothesis that GPCRs adopt functionally distinct conformations in a ligand-dependent manner to trigger differential downstream responses. In preliminary studies, specificity of sensor measurements was tested with b2-AR and led to the identification of a functional Gi conformation triggered by the beta-blocker metoprolol. Studies are structured to dissect distinct events in the GPCR-G protein interaction followed by stimulation with canonical and biased agonists. A clear understanding of the GPCR-G protein interaction and G protein selection from FRET/BRET studies is limited by the lack of control over relative concentration. Studies in aim 2 will leverage the SPASM technique to delineate the sequence of events prior to and after ligand stimulation. Key questions addressed are: (1) is the ligand-free GPCR basally associated or pre-coupled to a G protein? (2) Does the GPCR dissociate from the G protein following ligand-stimulation? (3) Does G protein selection correlate with the inherent binding affinity of a GPCR for a G protein? (4) To what extent is biased signaling influenced by the local concentration of GPCR and G protein? Systematic variation of the concentration of the GPCR-G protein interaction using the SPASM technique will be used to assess the specificity of FRET and downstream responses. The proposed research has the potential to create a new technical and conceptual platform for future studies on ligand-driven signaling initiated by any GPCR. The extensive validation of the SPASM sensors is an integral part of this study, which in turn will pave the way for their use in live-cell drug screens to identify small molecules that bias GPCR signaling towards therapeutically desired outcomes.