PTH and PTHrP Interaction with PTH Receptors

PTH 和 PTHrP 与 PTH 受体的相互作用

• 批准号: 7062731
• 负责人: THOMAS J GARDELLA
• 金额: $ 35.43万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062731
• 关键词: binding sites; circular dichroism; crosslink; gene mutation; hormone receptor; hormone regulation /control mechanism; nuclear magnetic resonance spectroscopy; osteoporosis; parathyroid hormone related protein; parathyroid hormones; peptide chemical synthesis; peptide hormone analog; protein protein interaction; protein structure function; receptor binding; site directed mutagenesis; tissue /cell culture

Description: (Taken directly from the application): Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) play critical roles in the regulation of blood mineral ion levels and organ development, respectively. They mediate these actions through the PTH/PTHrP receptor (PTH1R), a class 2 G protein-coupled receptor. A solid understanding of the molecular mechanisms by which these molecules interact and function, both in normal and disease states, is crucial to human health. Recent studies suggest that the (15-34) domain of PTH (and PTHrP ) "docks" to the N-terminal domain of the PTH1R and this high affinity interaction enables the PTH(1-14) domain to engage the heptahelical region of the receptor and thus induce receptor activation. But there are many unknowns. For example, the specific contacts that occur between the ligand and the receptor; the three-dimensional topologies of the ligand and receptor in the bound and free states, and the conformational changes that occur upon activation. These problems will be experimentally addressed in the proposed studies. We will apply "reductionist" approaches that expand on our prior work. We will use PTH(1-14) and PTH(15-34) analogs for structure-activity relationship analyses aimed at defining the ligand determinants of receptor activation and binding affinity, respectively. New types of amino acids and conformational constraints will be introduced into these ligands, some via our new collaboration with Dr. Greg Verdine of the Harvard University Chemistry Department, and. the resulting peptides will be used to functionally explore the PTH/PTH1R interaction mechanism. Thus, second-site suppression analyses will be performed with the ligands and mutant PTH1Rs altered by mutagenesis. As a complementary approach, photo-crosslinking studies will be performed in which photoderivatized PTH or PTHrP ligands are used to map sites of physical interaction with the receptor. We will also use intra-molecular, second-site suppression analyses, as well as divalent metal ion-chelation strategies to investigate the topology and dynamics of the heptahelical transmembrane domain region of the PTH1R. Our new collaborations with Dr. Mierke at Brown University will afford structural NMR analyses of the new ligand analogs and computer modeling of the PTHPTH1R complex. The overall studies will provide important new insights into the molecular mechanisms by which the PTH/PTHrP/PTH1R system mediates its crucial biological functions.

描述：（直接摘自申请）：甲状旁腺激素 (PTH) 和 PTH 相关肽 (PTHrP) 分别在调节血液矿物质离子水平和器官发育中发挥关键作用。它们通过 PTH/PTHrP 受体 (PTH1R)（一种 2 类 G 蛋白偶联受体）介导这些作用。深入了解这些分子在正常和疾病状态下相互作用和发挥作用的分子机制对于人类健康至关重要。最近的研究表明，PTH（和 PTHrP）的 (15-34) 结构域“对接”到 PTH1R 的 N 端结构域，这种高亲和力相互作用使 PTH(1-14) 结构域接合受体的七螺旋区域，从而诱导受体激活。但还有很多未知数。例如，配体和受体之间发生的特定接触；配体和受体在结合和游离状态下的三维拓扑结构，以及激活时发生的构象变化。这些问题将在拟议的研究中通过实验得到解决。我们将应用“还原论”方法来扩展我们之前的工作。我们将使用 PTH(1-14) 和 PTH(15-34) 类似物进行结构-活性关系分析，旨在分别确定受体激活和结合亲和力的配体决定因素。新型氨基酸和构象限制将被引入这些配体中，其中一些是通过我们与哈佛大学化学系的 Greg Verdine 博士的新合作实现的。所得肽将用于功能性探索 PTH/PTH1R 相互作用机制。因此，将使用通过诱变改变的配体和突变体 PTH1R 进行第二位点抑制分析。作为一种补充方法，将进行光交联研究，其中使用光衍生化 PTH 或 PTHrP 配体来绘制与受体物理相互作用的位点。我们还将使用 分子内第二位点抑制分析以及二价金属离子螯合策略来研究 PTH1R 七螺旋跨膜结构域区域的拓扑和动力学。我们与布朗大学 Mierke 博士的新合作将提供新配体类似物的结构 NMR 分析和 PTHPTH1R 复合物的计算机建模。总体研究将为 PTH/PTHrP/PTH1R 系统介导其关键生物学功能的分子机制提供重要的新见解。