The Research On The Interaction Between R-spondin And Its Receptors In Wnt Signaling

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.