Hormonal Control of Calcium Metabolism

总体说明（由申请人提供） 这个基础广泛的紧密整合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稳态的分子，细胞和生理作用。长期目标是了解PTH作用的细胞基础，这是其在骨质疏松症治疗价值的基础。项目一PTH和PTHrP与PTH受体的相互作用。PTH及其经典受体（PTHR 1）的活性生物分子复合物的分子结构的研究将进一步通过高效，强大的迭代测试方法，使用多种分子生物学和肽化学方法，结构上最小化配体和多个受体突变体。项目二. PTH/PTHrP受体的调节。将在体外和体内分析改变靶细胞上PTHR 1表面表达的正常发生的调节的生物化学和生理学后果，其中受体构建体经诱变以防止受体的磷酸化并损害受体的脱敏和内化。项目三.第二个消息在PTH行动。通过在体内施用信号选择性PTH配体，将探索由PTH刺激的PTHR 1的不同信号通路之间诱导选择性导致骨中独特的生物反应的证据。负责改变靶细胞反应的细胞机制将在体外用含有PTHR 1的信号选择性变体的骨细胞系来解决。项目四. PLC在PTH信号传导中的作用：体内突变受体。PLC依赖性PTHR 1信号传导的消除导致骨发育异常的证据，以及出生后骨结构完整性和矿物质离子稳态（特别是通过肾机制）的可诱导异常将通过用缺乏通过PLC途径的信号传导的遗传修饰的小鼠品系进行的广泛体内研究和用源自突变小鼠的细胞进行的基于机制的体外研究来探索。项目V. NHERF-介导的PTH信号在矿物质离子稳态。NHERF蛋白作为调节细胞信号传导的细胞内支架蛋白复合物的一部分起作用。NHERF最近被证明是作为“分子开关”，增加激活的PTH的PLC依赖性途径，同时抑制腺苷酸环化酶途径。NHERF蛋白的骨骼作用将在体内（使用最近报道的具有骨质减少的NHERF-1敲除动物）和体外用肾和骨细胞系研究，后者来自敲除动物和/或用NHERF蛋白转染。一个用于供应重要试剂的多功能肽核心和一个新建立的Skeleton表型核心，以及用于分析诱导骨骼反应的新工具，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.