The role of receptor complex composition and dynamics for signal specification and initiation in the WNT/Frizzled pathway

FrizzledFZD(FZD)家族的受体在胚胎发育和成年哺乳动物体内转导重要信号，FZD信号紊乱可导致从癌症和炎症性疾病到代谢和神经疾病等严重疾病。FZD受体(FZD_1~FZD_10)属于G蛋白偶联受体超家族，主要由分泌的WNT(Wingless/int1，WNT)家族脂糖蛋白激活。目前已鉴定出几条不同的WNT/FZD信号通路，它们分别是？连环蛋白依赖的信号通路和？连环蛋白非依赖的信号通路。到目前为止，WNT/FZD选择性或其他潜在机制如何定义途径选择性尚不清楚，是否受体复合体组成或与受体结合的WNT蛋白的功能选择性决定了下游信号转导，这个问题仍然悬而未决。假设FZD位于配体识别的中心，而辅助受体的招募可能将信号推向？连环蛋白依赖(LRP5/6)或？连环蛋白非依赖信号(ROR/RYK)信号。因此，本项目旨在分析受体复合体的组成和动力学，以及它们在WNT/FZD通路中的信号指定和启动中的重要性。两种不同的FZD模型蛋白(FZD4和FZD6)将被用来理解连接蛋白依赖(FZD4)和非依赖(FZD6)途径中的信号启动，特别是异源三聚体G蛋白的差异激活。为了了解信号指定的潜在机制，本项目主要关注获得受体组成和化学计量的准确信息。其次，利用FZD6受体分析了配体诱导的受体复合体动力学和在连接蛋白非依赖性途径中启动的信号转导。第三，将以FZD4为模型，评估辅助受体LRP6作为潜在的门卫在WNT诱导的连环蛋白或G蛋白依赖的通路之间切换信号的作用。FZD/共受体复合体内的蛋白质-蛋白质相互作用将通过生物化学、非天然氨基酸突变、活细胞成像结合Förster共振能量转移(FRET)、生物发光共振能量转移(BRET)和基于亲和力的光漂白后双色荧光(FRAP)方法进行分析。概述的项目旨在显著扩大对FZD和GPCR型受体生物学的总体了解。深入了解在这个重要的病理通讯系统中导致受体复合体激活和选择性信号启动的机制，将为开发针对Frizzled型受体的药物开辟新的可能性。

Receptors of the Frizzled (FZD) family transduce important signals during embryonic development and in adult mammals, and disturbed FZD signaling can cause severe diseases ranging from cancer and inflammatory diseases to metabolic and neurological disorders. Thus, FZDs present a promising though yet unexplored target for pharmaceutical intervention.FZD receptors (FZD1 to FZD10) belong to the superfamily of G protein-coupled receptors and are activated mainly by the secreted wingless/int1 (WNT) family lipoglycoproteins. Several distinct WNT/FZD-signaling pathways have been identified and they are distinguished as ß-catenin-dependent and ß-catenin-independent signaling routes. So far, it is unclear how WNT/FZD selectivity or other underlying mechanisms define pathway selectivity, and the question remains open, if receptor complex composition or the functional selectivity of the WNT protein that is binding the receptor determines downstream signaling. It is hypothesized, but yet unproven, that FZD is placed at the centre of ligand recognition, while the recruitment of co-receptors might push signaling towards either ß-catenin-dependent (LRP5/6) or ß-catenin-independent (ROR/RYK) signaling. Thus, this project aims to analyze receptor complex composition and dynamics, and their importance for signal specification and initiation in the WNT/FZD pathway. Two different FZD model proteins (FZD4 and FZD6) will be used to understand signal initiation in the ß-catenin-dependent (FZD4) and -independent (FZD6) pathway, with special attention to differential activation of heterotrimeric G proteins.To understand underlying mechanisms of signal specification, this project focuses primarily on gaining precise information of receptor composition and stoichiometry. Second, it is intended to analyze ligand-induced receptor complex dynamics and signaling initiated in the ß-catenin-independent pathway by using the FZD6 receptor. And third, the role of the co-receptor LRP6 as a potential gate keeper switching WNT-induced signaling between the ß-catenin- or G protein-dependent pathways will be evaluated employing FZD4 as a model. Protein-protein interactions within the FZD/co-receptor complex will be analyzed biochemically, as well as by unnatural amino acid mutagesesis, live cell imaging in combination with Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and by an affinity-based dual-color fluorescence after photobleaching (FRAP) approach.The outlined project aims at significantly expanding the knowledge on FZD and GPCR receptor biology in general. A thorough understanding of the mechanisms that lead to receptor complex activation and selective signal initiation in this pathologically important communication system will open novel possibilities for the development of drugs targeting class Frizzled receptors.