Functional Polarity of PTH Receptor Signaling: Cellular and Molecular Mechanisms

PTH 受体信号传导的功能极性：细胞和分子机制

• 批准号: 9380356
• 负责人: Peter A Friedman
• 金额: $ 61.16万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-20 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9380356
• 关键词: 25-hydroxyvitamin D; Adaptor Signaling Protein; Address; Affinity; Apical; Bilateral; Binding; Biochemical; Biological; Biomedical Research; Blood; CREB1 gene; CYP27B1 gene; Cell physiology; Cell surface; Cells; Chronic Kidney Failure; Clinical; Conflict (Psychology); Cyclic AMP; Data; Development; Disease; Down-Regulation; Endocrinology; Endosomes; Epithelial Cells; Equilibrium; Event; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; G-substrate; GTP-Binding Proteins; Generations; Genetic Transcription; Goals; Health; Homeostasis; Human; Hypophosphatemia; Ions; Kidney; Knockout Mice; Ligands; Link; Location; MDCK cell; Measures; Mediating; Medical; Metabolism; Microscopy; Minerals; Mixed Function Oxygenases; Modeling; Molecular; Mus; Outcome; PDZ protein; Parathyroid Hormone Receptor; Patients; Peptides; Phosphorylation; Physiological; Pilot Projects; Production; Property; Protein Hormone Receptor; Proteins; Proximal Kidney Tubules; Receptor Activation; Receptor Signaling; Renal tubule structure; Research; Role; Scaffolding Protein; Secondary Hyperparathyroidism; Serum; Signal Transduction; Signaling Protein; Specificity; Surface; Techniques; Testing; Therapeutic; Transactivation; Urine; Vitamin D; analog; apical membrane; base; basolateral membrane; biophysical tools; experimental study; in vivo; innovation; inorganic phosphate; kidney cell; mouse model; novel; parathyroid hormone-related protein; polarized cell; programs; receptor; receptor binding; receptor expression; receptor function; scaffold; sodium-hydrogen exchanger regulatory factor; stem; uptake

Project Summary The goal of this project is to determine the mechanisms underlying the molecular and cellular endocrinology of parathyroid hormone receptor (PTHR) signaling as it relates to phosphate and vitamin D balance. PTHR is uniquely expressed on both apical and basolateral membranes of the polarized cells that form renal proximal tubules. The biological consequences of bilateral PTHR expression or its origin are unknown. The premise of the proposal is that characterizing asymmetric PTHR actions will fill a major gap in our understanding of PTHR signaling and function and reconcile conflicting views of PTH action. Pilot studies show that PTHR signals from both basolateral and apical membranes but only basolateral PTHR activation induces transcription of the 1α-vitamin D hydroxylase (CYP27B1). Apical PTHR signaling primarily inhibits phosphate transport. The molecular and cellular mechanisms regulating PTHR actions are incompletely defined. The PTHR interacts at apical membranes with the PDZ scaffolding protein NHERF1, which tethers the receptor and regulates G-protein signaling and function. Absence of NHERF1 or its downregulation causes relocation of PTHR to basolateral membranes with an increased 1,25[OH]2D in mice and humans. Preliminary data show that Scribble, a basolateral PDZ protein, exerts a reciprocal effect, where downregulation causes accumulation of PTHR at apical membranes. We hypothesize that the polarized PTHR arrangement arises from the segregated location of NHERF1 and Scribble. We advance a research program to uncover new aspects of PTHR signaling in polarized kidney cells and test the central hypothesis that polarized PTHR expression is driven by the asymmetric location of the PDZ adaptor proteins, Scribble and NHERF, which in turn underlies the signaling bias of apical and basolateral PTHR actions on vitamin D and phosphate homeostasis. We propose three closely linked aims to evaluate this idea. The first two aims of address the hypothesis that basolateral PTHR activation preferentially stimulates the 1α-vitamin D hydroxylase, whereas apical PTHR signaling primarily blocks Pi transport. Aim 1 will determine the role of Scribble and NHERF in the generation of PTHR polarity and its asymmetric signaling in human kidney proximal tubule epithelial cells. Aim 2 uses live-cell FRET microscopy and other state-of-the art fluorescence techniques to characterize basolateral and apical membrane signaling properties of PTHR and the effect of NHERF1 and Scribble on biased G protein signaling. Aim 3 will delineate the in vivo actions of polarized proximal tubule PTHR by testing the role of Scribble on PTHR-dependent vitamin D and phosphate metabolism using a novel, conditional proximal tubule Scribble knockout mouse model that we generated. The outcomes will frame innovative therapeutic approaches targeting disorders of mineral metabolism.

项目概要 该项目的目标是确定分子和细胞内分泌学的潜在机制 甲状旁腺激素受体 (PTHR) 信号传导，因为它与磷酸盐和维生素 D 平衡相关。 在形成肾的极化细胞的顶膜和基底外侧膜上独特表达 双侧 PTHR 表达的生物学后果或其起源尚不清楚。 该提案的前提是，描述不对称 PTHR 行动的特征将填补我们的一个重大空白 了解 PTHR 信号传导和功能，并调和 PTH 作用的相互冲突的观点。 显示来自基底外侧膜和顶膜的 PTHR 信号，但仅基底外侧 PTHR 激活 诱导 1α-维生素 D 羟化酶 (CYP27B1) 的转录，主要抑制 Apical PTHR 信号传导。 调节 PTHR 作用的分子和细胞机制不完全。 PTHR 在顶膜与 PDZ 支架蛋白 NHERF1 相互作用，后者束缚。 受体并调节 G 蛋白信号传导和功能。 在小鼠和人类中，导致 PTHR 重新定位至基底外侧膜，并增加 1,25[OH]2D。 初步数据显示，基底外侧 PDZ 蛋白 Scribble 发挥着交互作用，其中 我们发现，下调会导致 PTHR 在顶膜处积累。 PTHR 排列源于 NHERF1 和 Scribble 的分离位置我们推进了一项研究。 计划揭示极化肾细胞中 PTHR 信号传导的新方面并测试中心假设 极化 PTHR 表达是由 PDZ 接头蛋白的不对称位置驱动的，Scribble 和 NHERF，这反过来又是顶端和基底外侧 PTHR 对维生素 D 作用的信号偏差的基础 我们提出了三个密切相关的目标来评估前两个目标。 解决了基底外侧 PTHR 激活优先刺激 1α-维生素 D 的假设 羟化酶，而顶端 PTHR 信号传导主要阻断 Pi 转运，Aim 1 将决定 PTHR 的作用。 Scribble 和 NHERF 在人肾中 PTHR 极性生成及其不对称信号传导中的作用 目标 2 使用活细胞 FRET 显微镜和其他最先进的技术。 荧光技术来表征 PTHR 的基底外侧和顶膜信号传导特性 NHERF1 和 Scribble 对偏向 G 蛋白信号传导的影响将描述 NHERF1 和 Scribble 的体内作用。 通过测试 Scribble 对 PTHR 依赖性维生素 D 的作用来极化近曲小管 PTHR 使用我们的新型条件性近曲小管 Scribble 敲除小鼠模型进行磷酸盐代谢 产生的结果将制定针对矿物质疾病的创新治疗方法。 代谢。