Molecular Basis of Wnt Receptor Interactions

说明(申请人提供)：WNTS是一种分泌型生长因子，在成人胚胎发育和组织更新过程中指定细胞命运。该通路的不适当激活与许多癌症有关。WNTs与两个共受体结合：7-跨膜螺旋受体称为FrizzledProteins(FRZ)，以及单程跨膜受体称为低密度脂蛋白受体相关蛋白5和6(LRP5/6)。LRP5或LRP6的激活导致其胞内区的磷酸化和Wnt信号的传递。WNT-FRZ-LRP5/6相互作用受多种激活剂和抑制剂的调节，包括脊椎动物的Dickkopf(DKK)蛋白。在这个建议中，生化、结构和生物物理分析被用来确定LRP5/6与Wnt途径激活剂和抑制剂相互作用的机制和特异性。这一结果将为未来在治疗上调节Wnt信号的努力提供一个机械基础。1.为了确定Dkk1如何调节LRP6的构象使其在Wnt信号中失活，将确定人Dkk1与LRP6不同部分结合的晶体结构，并将通过量热法准确地确定这些相互作用的能量。2.通过确定LRP6与激活剂(包括最近描述的单抗)结合的结构，以及通过确定LRP6不同区域对这些相互作用的能量贡献，来研究LRP6的激活状态。3.对通过结构突变确定为重要Dkk1相互作用残基的LRP6残基的突变进行检测，以与纯化的Wnt3a结合，以绘制LRP6上的Wnt3a结合表面。将对果蝇wntd进行结构研究，以确定对受体结合重要的同源经典wnts的表面。

DESCRIPTION (provided by applicant): Wnts are secreted growth factors that specify cell fate during embryogenesis and renewal of tissues in the adult. Inappropriate activation of the pathway is associated with a number of cancers. Wnts bind to two co- receptors: 7-transmembrane helix receptors called Frizzled proteins (Frz), and single pass transmembrane receptors called LDL-receptor related proteins 5 and 6 (Lrp5/6). Activation of Lrp5 or Lrp6 leads to phosphorylation of its intracellular domain and transmission of the Wnt signal. Wnt-Frz-Lrp5/6 interactions are modulated by various activators and inhibitors, including the vertebrate Dickkopf (Dkk) proteins. In this proposal, biochemical, structural, and biophysical analyses are used to define the mechanism and specificity of Lrp5/6 interactions with Wnt pathway activators and inhibitors. The results will provide a mechanistic underpinning for future efforts to modulate Wnt signaling therapeutically. 1. To define how Dkk1 modulates the conformation of Lrp6 to render it inactive in Wnt signaling, crystal structures of human Dkk1 bound different portions of Lrp6 will be determined, and the energetics of these interactions will be determined accurately by calorimetry. 2. The activated state of Lrp6 will be investigated by determining structures of Lrp6 bound to activators, including a recently described monoclonal antibody, and by determining energetic contributions of different regions of Lrp6 to these interactions. 3. Mutations of Lrp6 residues identified as important Dkk1 interaction residues by structure-based mutagenesis will be tested for binding to purified Wnt3a, in order to map out the Wnt3a binding surface on Lrp6. Structural studies will be performed on Drosophila WntD to define surfaces in the homologous classical Wnts important for receptor binding.