Hormonal Control of Calcium Metabolism

整体计划说明(由申请人提供) 这项基础广泛、紧密集成的研究计划旨在了解甲状旁腺激素(PTH)对骨和肾脏以及矿物质离子动态平衡的分子、细胞和生理作用。一个长期的目标是了解甲状旁腺素作用的细胞基础，这是甲状旁腺素治疗骨质疏松症的基础。项目1.甲状旁腺素和甲状旁腺素rP与甲状旁腺素受体的相互作用。甲状旁腺激素及其经典受体(PTHR1)的活性生物分子复合体的分子结构研究将通过高效、强大的迭代测试手段进一步进行，使用多种分子生物学和多肽化学方法，结构最小的配体和多个受体突变体。项目II.甲状旁腺素/甲状旁腺素受体的调节。将在体外和体内通过突变受体结构来分析改变靶细胞上正常发生的PTHR1表面表达调控的生化和生理后果，以防止磷酸化并损害受体的脱敏和内化。项目III.第PTH行动中的第二个信使有证据表明，PTHR1在PTH刺激的不同信号通路中的诱导选择性导致了骨中不同的生物学反应，这将通过体内注射信号选择性PTH配体来探索。负责改变靶细胞反应的细胞机制将在体外通过含有PTHR1信号选择性变体的骨细胞系来解决。项目IV：PLC在甲状旁腺激素信号转导中的作用：体内的突变受体。依赖PLC的PTHR1信号通路的异常会导致骨骼发育异常的证据，以及出生后骨骼结构完整性和可诱导的矿物质离子稳态异常(尤其是通过肾脏机制)的证据，将通过对缺乏PLC途径信号的转基因小鼠进行体内广泛研究，以及对来自突变小鼠的细胞进行体外基于机制的研究来探索。项目V.NHERF介导的PTH信号在矿物离子动态平衡中的作用NHERF蛋白是调节细胞信号的细胞内支架蛋白复合体的一部分。NHERF最近被证明是一个“分子开关”，通过PTH激活PLC依赖的通路，同时抑制腺苷环化酶通路。NHERF蛋白的骨骼作用将在体内(使用最近被报道有骨量减少的NHERF-1基因敲除动物)和体外肾脏和骨细胞系研究，后者来自基因敲除动物和/或转染NHERF蛋白。一个用于供应重要试剂的多功能多肽核心和一个新建立的具有诱导骨骼反应分析工具的骨骼表型核心为这五个项目提供了关键支持。

DESCRIPTION OF OVERALL PROGRAM (provided by applicant) This broad based tightly integrated research proposal is aimed at understanding the molecular, cellular and physiological actions of parathyroid hormone (PTH) on bone and kidney and mineral ion homeostasis. A long range goal is to understand the cellular basis of PTH actions which underlie its therapeutic value in osteoporosis. Project I. PTH and PTHrP Interaction with PTH Receptors. Study of the molecular structure of the active biomolecular complex of PTH and its classic receptor (PTHR1) will be pursued further by highly effective, powerful iterative testing approaches using multiple methods of molecular biology and peptide chemistry with structurally minimized ligands and multiple receptor mutants. Project II. Regulation of the PTH/PTHrP Receptor. The biochemical and physiological consequences of altering the normally occurring modulation of surface expression of PTHR1 on target cells will be analyzed in vitro and in vivo with receptor constructs mutagenized to prevent phosphorylation and impair desensitization and internalization of the receptor. Project III. Second Messengers in PTH Action. Evidence that induced selectivity among the diverse signalling pathways of PTHR1 stimulated by PTH lead to distinctive biological responses in bone will be explored by administration of signal-selective PTH ligands in vivo. Cellular mechanisms responsible for altered target cell response will be addressed in vitro with bone-cell lines containing signal selective variants of the PTHR1. Project IV. Role of PLC in PTH Signalling: Mutant Receptors in Vivo. Evidence that abrogation of PLC- dependent PTHR1 signalling leads to abnormalities in bone development, & post-natal bone structural integrity and inducible abnormalities in mineral ion homeostasis (especially through renal mechanisms) will be explored by extensive studies in vivo with a genetically modified mouse strain that lacks signaling through the PLC pathway and mechanism-based studies with cells derived from the mutant mice in vitro. Project V. NHERF- Mediated PTH Signalling in Mineral Ion Homeostasis. NHERF proteins function as part of intracellular scaffold protein complexes that modulate cellular signaling. NHERFs have recently been shown to act as "molecular switches" that increase activation by PTH of PLC-dependent pathways while suppressing adenyl cyclase pathways. The skeletal role of the NHERF proteins will be studied in vivo (using NHERF-1 knockout animals recently reported to have osteopenia) and in vitro with renal and bone cell lines, the latter derived from knockout animals and/or transfected with NHERF proteins. A versatile peptide core for supply of vital reagents and a newly established Skeletal Phenotyping Core with novel tools for analyses of induced skeletal responses provide critical support for the five projects.