Molecular Basis of Wnt Receptor Interactions

描述（由申请人提供）：Wnt是分泌的生长因子，在胚胎发生和成体组织更新期间指定细胞命运。该途径的不适当激活与许多癌症有关。Wnt与两种共受体结合：称为卷曲蛋白（Frz）的7跨膜螺旋受体和称为LDL受体相关蛋白5和6（Lrp 5/6）的单程跨膜受体。Lrp 5或Lrp 6的激活导致其细胞内结构域的磷酸化和Wnt信号的传递。Wnt-Frz-Lrp 5/6相互作用由各种激活剂和抑制剂调节，包括脊椎动物Dickkopf（Dkk）蛋白。在这个提议中，生化，结构和生物物理分析用于定义Lrp 5/6与Wnt通路激活剂和抑制剂相互作用的机制和特异性。这些结果将为将来治疗性调节Wnt信号转导提供机制基础。1.为了确定Dkk 1如何调节Lrp 6的构象以使其在Wnt信号传导中失活，将测定人Dkk 1结合的Lrp 6的不同部分的晶体结构，并且将通过量热法准确测定这些相互作用的能量。2. Lrp 6的激活状态将通过确定Lrp 6与激活剂结合的结构（包括最近描述的单克隆抗体）以及确定Lrp 6不同区域对这些相互作用的能量贡献来研究。3.将测试通过基于结构的诱变鉴定为重要Dkk 1相互作用残基的Lrp 6残基的突变与纯化Wnt 3a的结合，以绘制出Lrp 6上的Wnt 3a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Wnt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.