Functional Polarity of PTH Receptor Signaling: Cellular and Molecular Mechanisms

项目摘要 这个项目的目标是确定分子和细胞内分泌学的基础机制。 甲状旁腺激素受体(PTHR)信号转导与磷酸盐和维生素D平衡有关。PTHR 在形成肾脏的极化细胞的顶膜和基侧膜上都有独特的表达 近端小管。双侧PTHR表达的生物学后果或其来源尚不清楚。这个 该提案的前提是，表征不对称的PTHR行动将填补我们在 了解PTHR信号和功能，并调和对PTH作用的相互矛盾的观点。试点研究 显示PTHR信号来自基底侧膜和根尖膜，但仅来自基底外侧PTHR激活 诱导1-α-维生素D羟基酶(CYP27B1)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还不完全。 已定义。PTHR在顶膜与PDZ支架蛋白NHERF1相互作用，NHERF1系住 受体和调节G-蛋白信号和功能。NHERF1的缺失或其下调监管 导致PTHR在小鼠和人的基底侧膜上重新定位，1，25[OH]2D增加。 初步数据显示，底侧PDZ蛋白Scribble发挥了相互作用，其中 下调会导致PTHR在顶膜积聚。我们假设极化的 PTHR排列源于NHERF1和Scribble的分离位置。我们推进了一项研究 在极化肾细胞中发现PTHR信号的新方面并检验中心假说的计划 这种极化的PTHR表达是由PDZ接头蛋白的不对称位置Scribble驱动的 和NHERF，这反过来又是顶端和基底外侧PTHR作用于维生素D的信号偏向的基础 和磷酸盐动态平衡。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 解决了基底外侧甲状旁腺素受体激活优先刺激1α-维生素D的假说 羟基酶，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PTHR极性及其不对称信号产生中的作用 近端小管上皮细胞。AIM 2使用活细胞FRET显微镜和其他最先进的 荧光技术表征PTHR和PTHR的基侧和根尖膜信号特性 NHERF1和Scribble对偏向G蛋白信号转导的影响。目标3将描述在体内的作用 通过检测Scribble对PTHR依赖的维生素D和维生素D的作用来极化近端小管PTHR 使用一种新的、条件性近端小管Scribble基因敲除小鼠模型的磷酸盐代谢 已生成。这些结果将为针对矿物质紊乱的创新治疗方法奠定基础。 新陈代谢。

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.