Molecular Basis of Wnt Receptor Interactions

Wnt 受体相互作用的分子基础

• 批准号: 8441547
• 负责人: William I Weis
• 金额: $ 30.61万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8441547
• 关键词: Adult; Antibodies; Binding; Biochemical; C-terminal; Calorimetry; Cell surface; Cells; Chimera organism; Communication; Complex; Coupled; Development; Drosophila genus; EGF gene; Embryonic Development; Extracellular Domain; Fab Immunoglobulins; Family; Foundations; Future; Growth Factor; Human; LDL-Receptor Related Proteins; Length; Malignant Neoplasms; Maps; Molecular; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Mutation; Pathway interactions; Phosphorylation; Proteins; Regulation; Signal Transduction; Site-Directed Mutagenesis; Specific qualifier value; Specificity; Structure; Surface; Testing; Therapeutic Agents; Thermodynamics; Tissues; Titrations; Wnt proteins; base; defined contribution; inhibitor/antagonist; public health relevance; receptor; receptor binding; seven-transmembrane G-protein-coupled receptor; stem cell differentiation; stoichiometry; transmission process

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.

描述（由申请人提供）：Wnt 是分泌的生长因子，在胚胎发生和成人组织更新过程中指定细胞命运。该通路的不适当激活与许多癌症有关。 Wnt 与两个共受体结合：称为卷曲蛋白 (Frz) 的 7 次跨膜螺旋受体，以及称为 LDL 受体相关蛋白 5 和 6 (Lrp5/6) 的单次跨膜受体。 Lrp5 或 Lrp6 的激活导致其胞内结构域磷酸化并传输 Wnt 信号。 Wnt-Frz-Lrp5/6 相互作用受到各种激活剂和抑制剂的调节，包括脊椎动物 Dickkopf (Dkk) 蛋白。在该提案中，使用生化、结构和生物物理分析来定义 Lrp5/6 与 Wnt 通路激活剂和抑制剂相互作用的机制和特异性。这些结果将为未来调节 Wnt 信号传导的治疗提供机制基础。 1. 为了确定 Dkk1 如何调节 Lrp6 的构象以使其在 Wnt 信号传导中失活，将确定人 Dkk1 结合 Lrp6 不同部分的晶体结构，并通过量热法准确确定这些相互作用的能量。 2. 将通过确定与激活剂（包括最近描述的单克隆抗体）结合的 Lrp6 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 Lrp6 的激活状态。 3. 通过基于结构的诱变鉴定为重要的 Dkk1 相互作用残基的 Lrp6 残基的突变将测试其与纯化的 Wnt3a 的结合，以便绘制 Lrp6 上的 Wnt3a 结合表面。将对果蝇 WntD 进行结构研究，以确定对受体结合很重要的同源经典 Wnt 表面。