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道了不同的配体依赖的GPCR构象，但这些构象如何引起不同的下游反应仍不清楚。相反，从功能反应中破译G蛋白的选择是复杂的，因为有无数调节GPCR信号的因素，包括GPCRG蛋白的相对丰度和可用性，G蛋白在不同膜表面或微区的定位，以及调节蛋白如支架、激酶、阻滞素和细胞内吞机构的影响。这项拟议的研究通过PI开发的一种新型的基于FRET的痉挛传感器来解决这些限制，该传感器可以系统地调节活细胞中的蛋白质-蛋白质相互作用。研究将集中在四个GPCRs，b2-肾上腺素受体(b2-AR)，a2a-肾上腺素受体，黑素皮质素4受体和b3-肾上腺素受体，它们不同的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。AIM 1中的研究利用传感器来检测GPCR和来自GA亚单位c-末端的肽之间的相互作用，GA亚单位c-末端是GPCR-G蛋白配对的已知决定因素。基于FRET的测量将与既定的方法相结合，以检验GPCRs以配体依赖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，传感器测量的特异性用b2-AR进行了测试，并导致鉴定了由β-受体阻滞剂美托洛尔触发的功能性GI构象。研究的结构是剖析在GPCR-G蛋白相互作用中的不同事件，随后用规范的和有偏见的激动剂刺激。由于缺乏对相对浓度的控制，限制了对GPCR-G蛋白相互作用和从FRET/BRET研究中选择G蛋白的清楚了解。AIM 2的研究将利用痉挛技术来描述配体刺激前后的事件顺序。讨论的关键问题是：(1)无配体的GPCR是与G蛋白碱基结合还是预先偶联？(2)在配体刺激后，GPCR是否与G蛋白解离？(3)G蛋白的选择是否与G蛋白固有的结合亲和力相关？(4)GPCR和G蛋白的局部浓度在多大程度上影响有偏见的信号？利用痉挛技术对GPCR-G蛋白相互作用浓度的系统变化将被用来评估FRET及其下游反应的特异性。这项拟议的研究有可能为任何GPCR启动的配体驱动信号转导的未来研究创造一个新的技术和概念平台。对痉挛传感器的广泛验证是这项研究的组成部分，这反过来将为它们在活细胞药物筛选中的使用铺平道路，以识别将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.