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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要该项目的目的是确定分子和细胞内分泌学的机制与磷酸盐和维生素D平衡相关的甲状旁腺骑术受体（PTHR）信号传导的。 PTHR 在形成肾脏的偏振细胞的顶端和基底外侧膜上唯一表达近端管。双侧PTHR表达或其起源的生物学后果尚不清楚。该提案的前提是表征不对称的PTHR动作将填补我们的主要空白了解PTHR信号传导和功能，并调和PTH动作的冲突观点。试点研究证明来自基础膜和顶端膜的PTHR信号，但仅是Basolatorial PTHR激活诱导1α-Vitamin D羟化酶的转录（CYP27B1）。根尖PTHR信号传导主要抑制磷酸盐的运输。调节PTHR作用的分子和细胞机制不完全定义。 PTHR与PDZ脚手架蛋白NHERF1相互作用接收器并调节G蛋白信号传导和功能。缺乏NHERF1或其下调导致PTHR迁移到基底外侧膜中，而小鼠和人类中有1,25 [OH] 2D。初步数据表明，涂鸦是一种基础型PDZ蛋白，在其中执行互惠效果，其中下调导致PTHR在顶膜上的积累。我们假设两极分化 PTHR排列来自NHERF1和涂鸦的隔离位置。我们提出了一项研究计划在极化肾细胞中发现PTHR信号的新方面并检验中心假设极化的PTHR表达是由PDZ适配器蛋白的不对称位置驱动的和NHERF，这又是维生素D的顶端和基底外侧PTHR作用的信号传导偏置的基础和磷酸盐稳态。我们提出了三个紧密联系的目的，以评估这一想法。前两个目标解决基底外侧PTHR激活优先刺激1α-Vitamin D的假设羟化酶，顶端PTHR信号传导主要块PI转运。 AIM 1将决定 PTHR极性产生的涂鸦和NHERF及其在人肾脏中的不对称信号传导近端细胞上皮细胞。 AIM 2使用活细胞FRET显微镜和其他最新技术荧光技术表征PTHR和 NHERF1和涂鸦对偏置G蛋白信号传导的影响。 AIM 3将描述体内动作通过测试涂鸦对PTHR依赖性维生素D和使用一种新颖的条件近端细胞涂料敲除小鼠模型，我们的磷酸盐代谢生成。结果将构建针对矿物质疾病的创新治疗方法代谢。