Arthofer Elisa, Hot Belma, Petersen Julian, Strakova Katerina, Jäger Stefan, Grundmann Manuel, Kostenis Evi, Gutkind J Silvio, Schulte Gunnar
Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.)
Mol Pharmacol. 2016 Oct;90(4):447-59. doi: 10.1124/mol.116.104919. Epub 2016 Jul 25.
Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD6 assembles with Gαi1/Gαq (but not with Gαs, Gαo and Ga12/13), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD4, a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD4 assembles-in a DVL-independent manner-with Gα12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Gαi1, Gαo, Gαs, and Gαq The FZD4-G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD4 green fluorescent protein-transfected cells depend on Gα12/13 Furthermore, expression of FZD4 and Gα12 or Gα13 in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Gα12/13 signaling, underlining the functionality of an FZD4-Gα12/13-RHO signaling axis. In summary, Gα12/13-mediated WNT/FZD4 signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD4 signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.
卷曲蛋白（FZDs）是一类非传统的G蛋白偶联受体，属于卷曲蛋白家族。它们由分泌型脂糖蛋白的无翅/整合1（WNT）家族脂糖蛋白结合并激活。迄今为止，信号起始机制以及FZD与G蛋白的偶联仍知之甚少。此前，我们发现FZD6与Gαi1/Gαq组装在一起（但不与Gαs、Gαo和Ga12/13组装），并且这些非活性状态的复合物会被WNTs解离，并受磷蛋白散乱蛋白（DVL）调控。在此，我们研究了异源三聚体G蛋白与FZD4的非活性状态组装，FZD4是一种在视网膜血管发育中起重要作用的受体，在诺里病或家族性渗出性玻璃体视网膜病变中经常发生突变。使用光漂白后荧光恢复的活细胞成像实验表明，人FZD4以不依赖DVL的方式与Gα12/13组装在一起，但不与其他异源三聚体G蛋白亚家族的代表组装，如Gαi1、Gαo、Gαs和Gαq。FZD4-G蛋白复合物在受到WNT-3A、WNT-5A、WNT-7A和WNT-10B刺激后会解离。此外。WNT诱导的动态质量再分布变化在未转染细胞中存在，在FZD4绿色荧光蛋白转染的细胞中变化更明显，这依赖于Gα12/13。此外，在人胚肾293细胞中表达FZD4和Gα12或Gα13会诱导p115-RHOGEF（RHO鸟嘌呤核苷酸交换因子，分子量115 kDa）的WNT依赖性膜募集，p115-RHOGEF是Gα12/信号的直接靶点，这突出了FZD4-Gα12/13-RHO信号轴的功能。总之，通过p115-RHOGEF的Gα12/13介导的WNT/FZD4信号传导提供了一个有趣且此前未被认识到的机制联系，即FZD4信号传导与细胞骨架重排和RHO信号传导相关，这对胚胎发育和肿瘤发展过程中的血管生成调控具有重要意义。
