Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling
Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling
复制标题
DOI：
10.1016/j.gene.2006.12.014
复制
发表时间：
2007-04-15
期刊：
影响因子：
3.5
通讯作者：
Yaworsky, Paul J.
中科院分区：
文献类型：
--
作者：
Bhat, Bheem M.;Allen, Kristina M.;Yaworsky, Paul J.
在两个独立的家系中，低密度脂蛋白受体相关蛋白5(LRP5)基因的单点突变(G到T)导致了甘氨酸到缬氨酸的氨基酸变化(G171V)，并导致了常染色体显性高骨量性状(HBM)。LRP5作为与FrizzledWRITS家族成员的共同受体，传递可被LRP5配体Dickkopf I(Dkk1)拮抗的Wnt-规范信号。在WNT1存在的情况下，LRP5或HBM突变体(LRP5-G171V)诱导β-连环蛋白核转位并激活T细胞因子(TCF)-荧光素酶报告活性。HBM突变体抑制Dkk1功能，与LRP5相比，这导致对TCF活性的抑制减少。LRP5的结构分析表明，HBM突变位于第一个β-螺旋桨结构域的第4个叶片上。为了在体外阐明LRP5-G171V突变的功能意义和后果，我们采用基于结构的方法设计了15个特定的LRP5点突变。这些突变包括(A)叶片4中G171位的替换，(B)β-推进器1叶片2-6处的突变，以及(C)β-推进器2、3和4处的突变。在这里，我们证明了甘氨酸171位到K、F、I和Q的替换也导致了在Wnt1和Dkk1存在的情况下的HBM样活性。这表明G171位点的重要性，而不是特定氨基酸修饰对LRP5受体功能的影响。有趣的是，β-螺旋桨I的其他叶片(A65V、S127V、L200V、A214V和M282V)的G171等效残基突变导致了Dkk1功能的LRP5-G171V样阻断。然而，LRP5其他P推进器的G171V型突变并未导致对Dkk1功能的抵抗。这些结果表明LRP5β-螺旋桨I对Dkk1功能和WRIT信号的重要性。这些数据和对LRP5家族成员LDLR的额外比较结构分析表明，第一个β-螺旋桨结构域通过与Dkk1可以结合的LRP5的其他结构域的分子内相互作用发挥潜在的功能作用。这些研究还可能有助于更好地理解具有HBM样突变的LRP5的Dkk1样抑制配体功能降低的机制及其与骨密度表型增加的关系。(C)2006爱思唯尔B.V.保留所有权利。
A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Writs with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf I (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5-G171V) induces beta-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity. HBM variant suppresses Dkk1 function and this results in reduced inhibition of TCF activity as compared to that with LRP5. Structural analysis of LRP5 revealed that the HBM mutation lies in the 4th blade of the first beta-propeller domain. To elucidate the functional significance and consequence of the LRP5-G171V mutation in vitro, we took a structure-based approach to design 15 specific LRP5 point mutations. These included (a) substitutions at the G171 in blade 4, (b) mutations in blades 2-6 of beta-propeller 1, and (c) mutations in beta-propellers 2, 3 and 4. Here we show that substitutions of glycine at 171 to K, F, I and Q also resulted in HBM-like activity in the presence of Wnt1 and Dkk1. This indicates the importance of the G171 site rather than the effect of specific amino acid modification to LRP5 receptor function. Interestingly, G171 equivalent residue mutations in other blades of beta-propeller I (A65V, S127V, L200V, A214V and M282V) resulted in LRP5-G171V-like block of Dkk1 function. However G171V type mutations in other P-propellers of LRP5 did not result in resistance to Dkk1 function. These results indicate the importance of LRP5 beta-propeller I for Dkk1 function and Writ signaling. These data and additional comparative structural analysis of the LRP5 family member LDLR suggest a potential functional role of the first beta-propeller domain through intramolecular interaction with other domains of LRP5 wherein Dkk1 can bind. Such studies may also lead to a better understanding of the mechanisms underlying the reduced function of Dkk1-like inhibitory ligands of LRP5 with HBM-like mutations and its relationship to increased bone density phenotypes. (c) 2006 Elsevier B.V. All rights reserved.