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

受体复合物组成和动力学对 WNT/Frizzled 通路信号规范和启动的作用

• 批准号: 322725853
• 负责人: Dr. Maria Kowalski-Jahn
• 金额: --
• 依托单位: Institutionen för fysiologi och farmakologi
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/322725853?language=en
• 关键词: role; receptor; complex; composition; dynamics

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.

卷曲蛋白（FZD）家族的受体在胚胎发育期间和成年哺乳动物中提供重要信号，并且FZD信号传导紊乱可导致从癌症和炎性疾病到代谢和神经障碍的严重疾病。FZD受体（FZD 1 ~ FZD 10）属于G蛋白偶联受体超家族，主要由分泌的无翅/int 1（WNT）家族脂糖蛋白激活。已经鉴定了几种不同的WNT/FZD信号传导途径，并且它们被区分为β-连环蛋白依赖性和β-连环蛋白非依赖性信号传导途径。到目前为止，还不清楚WNT/FZD选择性或其他潜在机制如何定义途径选择性，并且如果受体复合物组成或结合受体的WNT蛋白的功能选择性决定下游信号传导，则问题仍然存在。假设但尚未证实的是，FZD位于配体识别的中心，而共受体的募集可能将信号传导推向β-连环蛋白依赖性（LRP 5/6）或β-连环蛋白非依赖性（ROR/RYK）信号传导。因此，本项目旨在分析受体复合物的组成和动力学，以及它们对WNT/FZD通路中信号特异性和起始的重要性。两种不同的FZD模型蛋白（FZD 4和FZD 6）将被用来了解信号启动的β-连环蛋白依赖（FZD 4）和β-连环蛋白不依赖（FZD 6）途径，特别关注的差异激活的异源三聚体G蛋白。为了了解信号规范的潜在机制，该项目主要集中在获得受体组成和化学计量的精确信息。其次，旨在通过使用FZD 6受体分析配体诱导的受体复合物动力学和在β-连环蛋白非依赖性途径中启动的信号传导。第三，将使用FZD 4作为模型来评估共受体LRP 6作为在β-连环蛋白或G蛋白依赖性途径之间切换WNT诱导的信号传导的潜在门控者的作用。FZD/辅助受体复合物内的蛋白质-蛋白质相互作用将进行生化分析，以及通过非天然氨基酸诱变，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）和基于亲和力的双色荧光光漂白（FRAP）概述的项目旨在大幅度扩展关于FZD和GPCR受体生物学的一般知识。深入了解导致受体复合物激活和选择性信号启动在这个病理上重要的通信系统的机制，将打开新的可能性，为开发药物靶向类卷曲受体。

项目成果

Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization

• DOI: 10.1074/jbc.ra118.004656
• 发表时间: 2018-10
• 期刊: The Journal of Biological Chemistry
• 作者: Kateřina Straková;Maria Kowalski-Jahn;Tomáš Gybeľ;J. Valnohova;V. Dhople;J. Harnoš;Ondřej Bernatík;R. Ganji;Z. Zdráhal;J. Mulder;C. Lindskog;V. Bryja;G. Schulte

Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment

• DOI: 10.1074/jbc.ra118.004763
• 发表时间: 2018-11-16
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Valnohova, Jana;Kowalski-Jahn, Maria;Schulte, Gunnar
• 通讯作者: Schulte, Gunnar