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: 9978053
• 负责人: Peter A Friedman
• 金额: $ 58.76万
• 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/9978053
• 关键词: 25-hydroxyvitamin D; Adaptor Signaling Protein; Address; Affinity; Agonist; 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; GTP-Binding Proteins; Generations; Genetic Transcription; Goals; Health; Homeostasis; Hormone Receptor Test; 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; Structure; 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.

项目摘要 本项目的目标是确定分子和细胞内分泌学的机制 甲状旁腺激素受体（PTHR）信号，因为它涉及磷酸盐和维生素D的平衡。PTHR 在形成肾脏的极化细胞的顶膜和基底膜上独特表达 近端小管双侧PTHR表达的生物学后果或其起源尚不清楚。的 该提案的前提是，描述不对称PTHR行动将填补我们在这方面的一个主要空白。 理解PTHR信号和功能，并调和PTH作用的冲突观点。试点研究 显示PTHR信号来自基底外侧膜和顶膜，但仅基底外侧PTHR激活 诱导1α-维生素D羟化酶（CYP 27 B1）的转录。心尖PTHR信号主要抑制 磷酸盐运输调节PTHR作用的分子和细胞机制尚不完全 定义了PTHR在顶端膜与PDZ支架蛋白NHERF 1相互作用， 受体和调节G蛋白信号和功能。NHERF 1缺失或下调 导致PTHR重新定位到基底外侧膜，在小鼠和人类中增加1，25 [OH]2D。 初步数据显示，基底外侧PDZ蛋白Scribble发挥了相互作用， 下调导致PTHR在顶膜处的积累。我们假设极化的 PTHR排列是由NHERF 1和Scribble的分离位置引起的。我们进行了一项研究 一项旨在揭示极化肾细胞中PTHR信号传导的新方面并检验中心假设的计划 极化PTHR表达是由PDZ衔接蛋白的不对称位置驱动的， 和NHERF，这反过来又是顶侧和基底侧PTHR对维生素D作用的信号偏差的基础 和磷酸盐平衡。我们提出了三个密切相关的目标来评估这一想法。前两个目标 基底外侧PTHR激活优先刺激1α-维生素D 羟化酶，而顶端PTHR信号主要阻断Pi转运。目标1将决定 Scribble和NHERF在人肾脏PTHR极性产生及其不对称信号转导中的作用 近曲小管上皮细胞目的2使用活细胞FRET显微镜和其他最先进的 荧光技术来表征PTHR的基底外侧和顶膜信号传导特性， NHERF 1和Scribble对偏向性G蛋白信号传导的影响。目的3将描述在体内的作用， 通过测试Scribble对PTHR依赖性维生素D的作用， 使用一种新的，条件性近端小管Scribble敲除小鼠模型， 生成的.这些结果将制定针对矿物质疾病的创新治疗方法 新陈代谢.

项目成果

Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

• DOI: 10.1073/pnas.2220851120
• 发表时间: 2023-06-06
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Stewart, Bryce Z.;Mamonova, Tatyana;Sneddon, W. Bruce;Javorsky, Airah;Yang, Yanmei;Wang, Bin;Nolin, Thomas D.;Humbert, Patrick O.;Friedman, Peter A.;Kvansakul, Marc
• 通讯作者: Kvansakul, Marc