PTH AND PTHRP INTERACTION WITH PTH RECEPTORS

PTH 和 PTHRP 与 PTH 受体的相互作用

• 批准号: 6300957
• 负责人: THOMAS J GARDELLA
• 金额: $ 22.9万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6300957
• 关键词: binding sites; crosslink; gene mutation; hormone receptor; hormone regulation /control mechanism; nuclear magnetic resonance spectroscopy; parathyroid hormone related protein; peptide chemical synthesis; peptide hormone analog; protein structure function; receptor binding; site directed mutagenesis; tissue /cell culture

Description:(Taken directly from the application) The critical role that parathyroid hormone (PTH) plays in the control of blood mineral ion levels, and the importance of PTH-related peptide (PTHrP) in skeletal development and hypercalcemia of malignancy, emphasize the need to understand the molecular mechanisms by which these ligands bind to and activate their receptors. Our prior studies suggest that the ligands binding domain "docks" to the N-terminal domain of the receptor, while the ligands N-terminal activation domain engages the receptor's "core" region. But the specifics are vague. The goals of this proposal are to define the molecular details of the ligand-receptor interface. We will approach the problem by generating and analyzing altered ligands and mutant receptors. Ligand residues will be analyzed in the context of small fragments corresponding to the activation domain, PTH(1-14), and the binding domain, PTH(17-31). This will permit scanning and saturation substitution approaches involving multiple peptides. For key substitutions, corresponding "intact" 1-34 or 1-31 peptides will be prepared for more extensive evaluations. Optimized activation and binding domains will be combined in "minimized" model peptides. The ligand work will be guided by NMR structural analyses, to be performed by our collaborators Drs. Weiss and Hua. In parallel to the ligand studies, we will define ligand-binding sites in the receptor using receptor mutagenesis. Second-site suppression analysis, where mutant receptors will be tested for intermolecular rescue of specifically modified PTH analogs, will be used to define point-to-point interactions. In this regard, we will define the molecular basis for ligand selectivity in the PTH-2 receptor, using mutant receptors and ligands modified at residues 5 and 23. To complement our functional studies, we will employ benzophenone (BPA)-containing ligands to cross-link ligand-receptor complexes. The topology and functional contribution of the seven helical domains of the receptor will be investigated using intramolecular second-site suppression analysis, and histidine-scanning strategies aimed to confer antagonist or agonist responsiveness to metal ions. As a longer-term goal, we explore the development of minimized soluble receptor fragments. The overall studies should provide important new information on the molecular determinants of ligand recognition and the mechanisms of ligand-induced receptor activation in PTH receptor systems.

描述：（直接摘自申请）甲状旁腺激素（PTH）在控制血液矿物质离子水平中的关键作用，以及PTH相关肽（PTHrP）在骨骼发育和恶性肿瘤高钙血症中的重要性，强调需要了解这些配体结合并激活其受体的分子机制。我们先前的研究表明，配体结合结构域“对接”到受体的N-末端结构域，而配体N-末端活化结构域接合受体的“核心”区域。但具体细节还不清楚。该提案的目标是定义配体-受体界面的分子细节。我们将通过产生和分析改变的配体和突变的受体来解决这个问题。将在对应于活化结构域PTH（1-14）和结合结构域PTH（17-31）的小片段的背景下分析配体残基。这将允许涉及多个肽的扫描和饱和取代方法。对于关键置换，将制备相应的“完整”1-34或1-31肽用于更广泛的评价。优化的活化和结合结构域将在“最小化”模型肽中组合。配体工作将由我们的合作者韦斯博士和华博士进行NMR结构分析指导。与配体研究平行，我们将使用受体诱变来定义受体中的配体结合位点。第二个网站的抑制分析，突变受体将被测试为分子间救援的具体修改PTH类似物，将被用来定义点对点的相互作用。在这方面，我们将定义的配体选择性的PTH-2受体的分子基础，使用突变体受体和配体在残基5和23修改。为了补充我们的功能研究，我们将采用二苯甲酮（BPA）的配体交联配体-受体复合物。拓扑结构和功能的贡献的受体的七个螺旋结构域将使用分子内第二个网站的抑制分析，和组氨酸扫描策略，旨在赋予拮抗剂或激动剂的反应，金属离子。作为一个长期的目标，我们探索最小化可溶性受体片段的发展。这些研究将为PTH受体系统中配体识别的分子决定因素和配体诱导受体活化的机制提供重要的新信息。