PTH and PTHrP Interaction with PTH Receptors

描述：(直接取自本申请)：甲状旁腺激素(PTH)和甲状旁腺激素相关肽(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)介导这些作用，PTH1R是一种2G蛋白偶联受体。对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有一个坚实的理解，这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PTH1R的N-末端结构域，这种高亲和力相互作用使PTH(1-14) 与受体的七螺旋区域结合，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自由状态下的三维拓扑结构，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以实验方式解决。我们将采用在先前工作基础上扩展的“简化论”方法。我们将使用PTH(1-14)和PTH(15-34)类似物进行构效关系分析，旨在分别确定受体激活和结合亲和力的配体决定因素。新类型的氨基酸和构象限制将被引入到这些配体中，一些是通过我们与哈佛大学化学系的Greg Verdine博士的新合作，以及。得到的多肽将用于从功能上探索PTH/PTH1R相互作用的机制。因此，将对通过突变改变的配体和突变的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，其中使用光衍生化的PTH或PTHrP配体来绘制与受体的物理相互作用的位置。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，以研究PTH1R的七螺旋跨膜区的拓扑和动力学。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核磁共振分析和PTHPTH1R络合物的计算机模拟。这些研究将为甲状旁腺素/甲状旁腺素相关蛋白/甲状旁腺素1R系统调节其重要生物学功能的分子机制提供重要的新见解。

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.