【作者】 徐扬；

【导师】 施维；

【作者基本信息】 吉林大学 ， 细胞生物学， 2013， 博士

【摘要】 Wnt信号通路是依赖于细胞分泌蛋白Wnt与其细胞表面受体结合而被激活的重要细胞通路。自发现至今已有三十余年。Wnt通路根据其是否依赖β-cantenin，而被分为经典Wnt通路和非经典Wnt通路。经典Wnt通路由胞外Wnt配体同时结合一个七次跨膜受体（Frizzled）和低密度脂蛋白受体相关蛋白5/6（Lrp5/6）而被激活。被Wnt蛋白激活的受体Frizzled和Lrp6，将抑制胞内Wnt通路效应物β-catenin的降解，从而激活下游通路。经典Wnt通路（以下简称Wnt通路）在生物体的胚胎形成和发育以及干细胞的更新和分化中起着重要作用。此外，近年来的研究发现，Wnt通路的异常与肿瘤和骨质酥松等疾病也有很大关系。因此，开展对Wnt通路的深入研究，将有利于探索治疗上述疾病的新方法。在Wnt通路中，尚存在着多种胞外或者胞内的拮抗剂和激动剂，共同调节Wnt通路的活性。R-spondin便是Wnt通路最重要的激动剂之一。它在决定动物性别、介导胚胎四肢形成、影响胚胎血管形成和指甲发育等方面具有重要作用。尽管R-spondin对Wnt通路的激活作用已经确定，但其受体一直存在争议。Lrp6、Frizzled和Kremen，曾被认为是R-spondin的受体。然而，最近几年的研究结果表明，七次跨膜蛋白LGR4/5/6和单次跨膜蛋白ZNRF3/RNF43是R-spondin最可能的受体。最初曾认为，LGR是通过R-spondin分别与Lrp、Wnt以及Frizzled形成复合物而激活下游Wnt通路的。直到发现了R-spondin受体ZNRF3/RNF43参与Wnt信号通路之后，R-spondin激活Wnt通路的模型才得到了更新。最新研究结果表明，当R-spondin不存在时，E3泛素连接酶ZNRF3/RNF43持续泛素化Wnt受体Frizzled，使Frizzled被降解，从而抑制了Wnt信号通路；而当R-spondin存在时，R-spondin与LGR和ZNRF3/RNF43结合形成复合物，进而将ZNRF3/RNF43拽离Frizzled，Frizzled的降解被抑制，蛋白水平上升，Wnt通路被间接激活。虽然R-spondin的受体陆续被发现，但这些受体行使功能的机理以及与R-spondin相互作用的方式，仍然存在争议。这正是本领域目前研究的热点之一。对此，本论文着重研究了R-spondin及其受体LGR4/5和ZNRF3/RNF43在Wnt通路中的作用机理，达到了预期的研究目标，取得了如下的研究成果：首先，本论文着重研究了R-spondin受体LGR4/5激活Wnt通路的机理。依据“Wnt通路的两个拮抗剂DKK1和SFRP1可分别与受体Lrp6和Frizzled5的胞外区相互作用”这一特性，构建了串联蛋白DKK1-LGR4/5和SFRP1-LGR4/5，探索了LGR4/5是否通过与Lrp6或Frizzled5结合而激活下游Wnt通路的机理。结果表明，DKK1-LGR4可以帮助Lrp6激活Wnt通路，但其协同能力不强；而DKK1-LGR5则难以协助Lrp6激活Wnt通路；SFRP1-LGR4/5和Frizzled5共同转染可以激活Wnt通路，这主要是串联蛋白中的SFRP1将细胞自身分泌的Wnt3a富集到Frizzled5附近所致。虽然LGR4和Lrp6接触，可一定程度地激活Wnt通路，但LGR4和LGR5激活Wnt通路的主要方式，并不完全是通过与Lrp6或Frizzled5的接触，尚需其他蛋白的协助和参与。其次，本论文探讨了R-spondin1和Wnt3a的相互作用及其domain mapping（确认作用区域）。人类有19种Wnt，但其分别与R-spondin的协同效果并不清楚。通过Top-Flash reporter（检测Wnt通路是否被激活的荧光素酶报告实验，即luciferase）实验，检测了19种Wnt在不同浓度时单独激活Wnt通路的能力及其与R-spondin1的协同效果。结果表明，在19种Wnt中，Wnt1和Wnt3a分别单独激活Wnt通路的能力最强，而且与R-spondin1具有较好的协同效应。随后，实验选取了其中被广泛使用且效果较强的Wnt3a，探讨了其与R-spondin1的相互作用关系。根据序列比对和结构分析，更加科学地定义了R-spondin的不同结构域，通过分子克隆手段，成功地构建了R-spondin1的6种结构域缺失突变体（Furin-myc、Tsp-myc、△Furin1-myc、△Furin2-myc、Furin1-myc和Furin2-myc），进而研究了R-spondin1和Wnt3a的相互作用及其domain mapping。结果显示，R-spondin1和Wnt3a之间存在相互作用；R-spondin1的Furin1结构域主要负责与Wnt3a的结合。luciferase的结果显示，Furin1结构域单独与Wnt3a并没有协同作用，这说明R-spondin1尚需其他结构域与其他R-spondin受体的协助作用，才能完成R-spondin协同Wnt3a的功能。此外，实验还构建了4种R-spondin的点突变体（Q71R、N51S、Y83A和G73R），探讨了Furin1结构域的特殊位点对R-spondin1功能的影响。得出的结论是：Furin1结构域的73位的甘氨酸和83位的酪氨酸对R-spondin1的活性影响较大。最后，本论文就R-spondin及其受体LGR4和ZNRF3/RNF43的相互作用以及domain mapping进行了研究。结果表明，R-spondin1分别与LGR4和ZNRF3/RNF43间均存在着较强的相互作用；其中Furin2结构域主要负责R-spondin1与LGR4的结合，而Furin1结构域主要负责与RNF43的相互作用；并且R-spondin1的Tsp结构域与ZNRF3间可能存在一定的相互作用。同时，我们还检测了R-spondin2的Furin结构域、Furin1和Furin2结构域分别与ZNRF3和RNF43的相互作用，结果发现，Furin结构域分别与RNF43和ZNRF3之间均存在着较强的相互作用关系，而R-spondin1的Furin结构域与ZNRF3的作用很弱，说明R-spondin1和R-spondin2分别与受体间的作用存在细微差异。免疫共沉淀的结果还显示，ZNRF3和RNF43分别与其配体的作用存在差异，其中R-spondin2的Furin1结构域与RNF43的相互作用更强。同时发现，ZNRF3/RNF43与Frizzled8胞外区的相互作用较弱，尚需胞内区和（或者）跨膜区的协助和参与。luciferase实验证实，ZNRF3和RNF43可抑制R-spondin激活Wnt通路；R-spondin2的Furin结构域与Wnt3a具有较强的协同效果；而Furin1结构域单独对Wnt通路作用时却没有活性，这与R-spondin1的Furin1表现一致。通过上述研究，基本确定了R-spondin分别与Wnt3a、LGR4以及ZNRF3/RNF43相互作用机制。这为进一步研究R-spondin激活Wnt通路的机理奠定了基础。同时，为Wnt通路相关疾病的治疗和药物靶点的探索提供了新线索。更多还原

【Abstract】 Wnt signaling is an important cell signaling whose activation is dependent on thebinding between Wnt protein and its receptor on the cell surface. Three decades havebeen passed since its discovery. Wnt signaling is divided into two kinds according towhether it needs β-cantenin. One is canonical Wnt signaling, the other isnon-canonical Wnt signaling. When Wnt ligands bind to both theseven-transmembrane receptor (Frizzled) and a low density lipoproteinreceptor-related protein5/6(Lrp5/6), the canonical Wnt signaling will be turned on.This binding will lead to the inhibition of β-cantenin’s degradation, resulting in theactivation of Wnt signaling. The canonical Wnt signaling (term as “Wnt signaling”below) has an important role in formation and development of embryos, and also inself-renew and differentiation of stem cells. Besides, the abnormity of Wnt signalingis found related to some diseases, such as cancer, osteoporosis and so on, according tothe recent studies. Thus, it is of great benefit for finding new treatments against thediseases mentioned above with making an intensive study of Wnt pathway.There are lots of extracellular and intracellular Wnt antagonists and agonists inWnt pathway. They take charge of regulating the activity of Wnt pathway. R-spondinis one important member of the agonists. It has important roles on genderdetermination, mediating limb formation, affecting embryonic blood vessel formationand nail development. Although the positive role of R-spondin in Wnt signaling hasalready been confirmed, its receptor is still in dispute. Lrp6, Frizzled and Kremenwere considered as the receptors of R-spondin. However, the current findings showthat seven-transmembrane protein LGR4/5/6and single transmembrane receptorZNRF3/RNF43are now considered as receptors of R-spondin.LGR was initially considered to bind with R-spondin and Lrp6. They form the complex together with Wnt and Frizzled to activating Wnt pathway. The model ofhow R-spondin activates the pathway was renewed after the discovery of thatZNRF3/RNF43involve in Wnt signaling as R-spondin’s receptor. The latest researchshows that, in the absent of R-spondin, E3ubiquitin ligase ZNRF3/RNF43willconstantly ubiquitinate Wnt receptor, Frizzled, leading to the degradation of Frizzled,which inhibits Wnt pathway. While R-spondin appears, R-spondin, LGR andZNRF3/RNF43will form a complex, resulting in the departure of ZNRF3/RNF43from Frizzled. The degradation of Frizzled will then be restrained. As a result, theprotein level of Frizzled rises up and Wnt signaling is enhanced.Although the receptors of R-sponidn are found one after another, the underlyingaction mechanism of R-spondin and its receptors is still a hot pot of this area. Thus,this study focused on the detailed action mechanism of R-spondin and its receptors,LGR4/5and ZNRF3/RNF43; reached expected target; achieved the following results.Firstly, the mechanism of how LGR4/5triggers Wnt pathway was studied. Wemade two kinds of series proteins DKK1-LGR4/5and SFRP1-LGR4/5using thetheory that Wnt antagonists DKK1and SFRP1can bind to Lrp6and Frizzled5respectively. Then we used these two series proteins to explore whether LGR4/5activate the downstream via binding with Lrp6or Frizzled5. The results showed thatDKK1-LGR4could synergize with Lrp6, which is not very prominent. WhileDKK1-LGR5could not. SFRP1-LGR4/5could synergize with Frizzled5because ofthe SFRP1part in SFRP1-LGR4/5which enriched the endogenous Wnt3a toFrizzled5. Although the association of LGR4with Lrp6could slightly enhance thesignal, the main manner in which LGR4/5activates the Wnt pathway may not rely onthe binding with Lrp6or Frizzled5. Other proteins involved should be needed.Secondly, this study probed into the way that R-spondin1binds with Wnt3a, andtheir domain mapping. There are19kinds of Wnt proteins in Human, but theirsynergism abilities with R-spondin are unclear. By Top-Flash reporter assay(Luciferase experiment which detects the level of Wnt pathway activation.), theabilities of activating the Wnt pathway by19kinds of Wnt at different concentrationalone and the synergism between19kinds of Wnts and R-spondin1were detected. The data showed that among the19Wnts, Wnt1and Wnt3a each had the strongestability of enhancing Wnt signaling and had better synergism with R-spondin1. Soonafterwards, Wnt3a was chosen to study the interaction with R-spondin1because it iswidely used and has strong effect. According to sequence alignment and structuralanalysis, different domains of R-spondin were defined more scientifically. By usingmolecular cloning,6domain deletion mutants (Furin-myc, Tsp-myc,△Furin1-myc,△Furin2-myc, Furin1-myc and Furin2-myc) were successfully made. Then theinteraction between R-spondin1and Wnt3a, the domain mapping of the interactionwere tested. The data showed that R-spondin1associated with Wnt3a. Furin1domainof R-spondin1took charge of the binding with Wnt3a. Top-Flash reporter assayshowed that Furin1could not synergize with Wnt3a functionally, which suggested thatother domains of R-spondin1might be needed to enhance Wnt pathway by binding toother receptors. Besides, four point mutants of R-spondin1(Q71R, N51S, Y83A andG73R) were cloned and used to explore the function of these amino acid sites. Theconclusion is that73glycine and83tyrosine are important for R-spondin1’s activity.Finally, this thesis studied the binding between R-spondin1and its receptors,LGR4and ZNRF3/RNF43, and their domain mapping. The results indicated that therewere strong binding between R-spondin1and its receptors. Furin2domain bound withLGR4, while Furin1domain bound with RNF43, and Tsp domain might interact withZNRF3. Meanwhile, we detected the interaction between Furin domain, Furin1domain, Furin2domain of R-spondin2and ZNRF3/RNF43separately. The resultsshowed that Furin domain had strong binding with both RNF43and ZNRF3; whilethe association between Furin of R-spondin1and ZNRF3was weak, which suggestedthat there is nuance between the interaction of R-spondin1and R-spondin2with theirreceptors. The co-immunoprecipitation data indicated that the ways of interaction ofZNRF3and RNF43with their ligands were also distinct, as RNF43bound better withFurin2of R-spondin2. At the same time, we found that ZNRF3/RNF43had weakinteraction with Frizzle8extracellular region, which illustrated that this binding mightneed the help of the cytoplasmic regions and/or transmembrane domains. Luciferaseexperiments showed that ZNRF3and RNF43could inhibit Wnt pathway enhanced by R-spondin; the Furin domain of R-spondin2synergized with Wnt3a well; Furin1domain of R-spondin2had no effect on Wnt3a, which is consistent with the behaviourof R-spondin1’s Furin1domain.Based on the study above, we confirmed the main mechanism of the associationbetween R-spondin and Wnt3a, LGR4/5, ZNRF3/RNF43. This lays a foundation forthe further study of the binding mechanism, and provides some clues for finding newtreatment and drug target.更多还原