Molecular Basis of Wnt Receptor Interactions

描述（由申请人提供）：wnt是一种分泌的生长因子，在胚胎发生和成人组织更新过程中指定细胞命运。该通路的不当激活与许多癌症有关。wnt结合两种共受体：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 （Lrp5/6）的单通道跨膜受体。Lrp5或Lrp6的激活导致其胞内结构域的磷酸化和Wnt信号的传递。Wnt-Frz-Lrp5/6相互作用受多种激活剂和抑制剂调节，包括脊椎动物Dickkopf （Dkk）蛋白。本研究采用生化、结构和生物物理分析来确定Lrp5/6与Wnt通路激活剂和抑制剂相互作用的机制和特异性。该结果将为未来的治疗性调节Wnt信号提供机制基础。1. 为了确定Dkk1如何调节Lrp6的构象，使其在Wnt信号传导中失活，我们将确定人类Dkk1结合Lrp6不同部分的晶体结构，并通过量热法精确确定这些相互作用的能量学。2. Lrp6的激活状态将通过确定Lrp6与激活剂（包括最近描述的单克隆抗体）结合的结构，并通过确定Lrp6不同区域对这些相互作用的能量贡献来研究。3. 通过结构诱变鉴定为重要的Dkk1相互作用残基的Lrp6残基突变将被测试与纯化的Wnt3a结合，从而绘制出Wnt3a在Lrp6上的结合表面。结构研究将在果蝇WntD上进行，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. PUBLIC HEALTH RELEVANCE: Wnt proteins are secreted growth factors that are essential for the differentiation of stem cells. This proposal aims to understand how Wnts interact with their receptors on the cell surface. The molecular information from these studies could provide a foundation for rational, efficient development of therapeutic agents to control inappropriate Wnt signaling in a number of cancers.