PTH AND PTHRP INTERACTION WITH PTH RECEPTORS

描述：（直接从申请中获取）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。我们之前的研究表明，配体结合域与受体的n端结构域“对接”，而配体n端激活域与受体的“核心”区域接合。但具体细节尚不明确。本建议的目标是定义配体-受体界面的分子细节。我们将通过生成和分析改变的配体和突变受体来解决这个问题。配体残基将在激活域PTH（1-14）和结合域PTH（17-31）对应的小片段的背景下进行分析。这将允许扫描和饱和取代方法涉及多个多肽。对于关键替换，将制备相应的“完整的”1-34或1-31肽以进行更广泛的评价。优化的激活和结合结构域将结合在“最小化”的模型肽中。配体工作将由核磁共振结构分析指导，由我们的合作者博士。魏斯和华。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。第二位点抑制分析，其中突变受体将测试特异性修饰的PTH类似物的分子间拯救，将用于定义点对点相互作用。在这方面，我们将定义PTH-2受体中配体选择性的分子基础，使用突变受体和在残基5和23处修饰的配体。为了补充我们的功能研究，我们将使用含二苯甲酮（BPA）的配体来交联配体-受体复合物。受体的七个螺旋结构域的拓扑结构和功能贡献将通过分子内第二位点抑制分析和组氨酸扫描策略来研究，目的是赋予拮抗剂或激动剂对金属离子的反应性。作为一个长期的目标，我们探索开发最小的可溶性受体片段。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的受体激活机制提供重要的新信息。

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.