Probing molecular mechanisms of GPCR functional selectivity in live cells

描述（由申请人提供）：虽然最近有几项研究报道了不同的配体依赖性GPCR构象，但尚不清楚这些构象如何引起不同的下游反应。相比之下，从功能反应中解读G蛋白的选择是复杂的，因为调控GPCR信号传导的无数因素，包括GPCR的相对丰度和可用性、G蛋白、不同膜表面或微域的定位，以及调控蛋白（如支架、激酶、抑制蛋白和细胞内吞装置）的影响。提出的研究通过PI开发的一类新型基于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。研究将集中于4种gpcr， b2-肾上腺素受体（b2-AR）， a2a -肾上腺素受体，黑素皮质素受体和b3-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影响。目标1中的研究利用传感器检测GPCR与Ga亚基c端衍生的肽之间的相互作用，这是GPCR- g蛋白配对的已知决定因素。基于fret的测量将与已建立的方法相结合，以验证gpcr以依赖配体的方式采用功能不同的构象来触发不同的下游反应的假设。在初步研究中，用b2-AR测试了传感器测量的特异性，并鉴定了β受体阻滞剂美托洛尔触发的功能性Gi构象。研究的结构是剖析GPCR-G蛋白相互作用中的不同事件，随后是规范和偏倚激动剂的刺激。由于缺乏对相对浓度的控制，从FRET/BRET研究中对GPCR-G蛋白相互作用和G蛋白选择的清晰理解受到限制。目标2的研究将利用SPASM技术来描述配体刺激前后的事件序列。解决的关键问题是：(1)无配体GPCR是与G蛋白基本相关还是预偶联？(2) GPCR在配体刺激下是否与G蛋白分离？(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)局部GPCR和G蛋白浓度对偏态信号的影响程度如何？使用SPASM技术对GPCR-G蛋白相互作用浓度的系统变化将用于评估FRET和下游反应的特异性。提出的研究有可能为任何GPCR启动的配体驱动信号的未来研究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，以识别偏向GPCR信号的小分子，以达到治疗所需的结果。

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.