PTH and PTHrP Interaction with PTH Receptors

PTH 和 PTHrP 与 PTH 受体的相互作用

• 批准号: 7160504
• 负责人: THOMAS J GARDELLA
• 金额: $ 34.28万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7160504
• 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.

产品描述：（直接摘自应用）：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起着关键作用。它们通过PTH/PTHrP受体（PTH 1 R）（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PTH 1 R的N-末端结构域，这种高亲和力的相互作用使PTH（1-14） 结构域以接合受体的七螺旋区域并因此诱导受体活化。但还有很多未知数。例如，发生在配体和受体之间的特异性接触;在结合和游离状态下配体和受体的三维拓扑结构，以及在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加以解决。我们将应用“简化”的方法，扩展我们以前的工作。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，旨在分别确定受体活化和结合亲和力的配体决定因素。新型的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。所得肽将用于功能性地探索PTH/PTH 1 R相互作用机制。因此，第二位点抑制分析将用通过诱变改变的配体和突变PTH 1 R进行。作为补充方法，将进行光交联研究，其中使用光衍生的PTH或PTHrP配体来绘制与受体的物理相互作用位点。我们还将使用 分子内，第二个网站的抑制分析，以及二价金属离子螯合的策略，以调查的拓扑结构和动力学的七螺旋跨膜结构域区域的PTH 1 R。我们与布朗大学的Mierke博士的新合作将提供新配体类似物的结构NMR分析和PTHPTH 1 R复合物的计算机建模。这些研究将为PTH/PTHrP/PTH 1 R系统介导其重要生物学功能的分子机制提供重要的新见解。