Effects of NHERF1 expression on PTH1R dynamics and function in polarized cells

描述(由申请人提供)：G蛋白偶联受体需要胞质适配器蛋白才能正常发挥功能和调节。一个很好的例子是NaVhT交换调节因子1(NHERF1)对1型甲状旁腺激素受体(PTH1R)的调节。在非极化细胞中，NHERF1已被证明改变了PTH1R的横向迁移率、信号和内化。在这项研究中，我们将研究NHERF1在极化近曲小管(PCT)细胞PTH1R功能调节中的作用。在PCT细胞中，PTH1R在顶膜和基底膜上均有表达，而NHERF1仅在顶膜上表达。由于NHERF1的这种极化表达，我们假设PTH1R在两种膜上的行为将不同。NHERF1对心尖和基底外侧受体的分布、移动、信号和内化的影响将被研究。我们预测，NHERF1会将PTH1R锚定在顶端的肌动蛋白细胞骨架上，导致细胞骨架的侧向迁移率和内化率降低。此外，这种拴系有望导致配体诱导的钙瞬变。NHERF1基因敲除锚定的中断，或无法结合NHERF1或不结合细胞骨架的NHERF1突变体的受体结构的表达，有望逆转NHERF1的极化效应，并导致整个细胞统一的PTH1R行为。这些研究将使用各种活细胞成像技术，如光漂白后的荧光恢复、量子点单分子跟踪、cAMP和钙的光学测量以及图像互相关分析。这些研究将加深我们对NHERF1在近曲小管生理相关环境中对PTH1R调节的理解。由于近端小管中PTH1R和NHERF1的紊乱导致磷酸盐稳态的破坏，对它们的生理学的更好的理解可能会导致新的治疗策略。由于NHERF1与广泛的GPCRs、离子通道和酪氨酸激酶相互作用，这些研究的扩展可以转化为对许多疾病的治疗。

DESCRIPTION (provided by applicant): G-protein coupled receptors require cytosolic adaptor proteins for proper function and regulation. A prime example is the regulation of type 1 parathyroid hormone receptor (PTH1R) by NaVhT Exchange Regulatory Factor 1 (NHERF1). In non-polarized cells, NHERF1 has been shown to alter the lateral mobility, signaling and internalization of the PTH1R. In this proposal we will investigate the role of NHERF1 in the regulation of PTH1R function in polarized proximal convoluted tubule (PCT) cells. In PCT cells PTH1R is expressed on both the apical and basolateral membrane while NHERF1 localization is confined to the apical membrane. Because of this polarized expression of NHERF1, we hypothesize that PTH1R will behave differently in the two membranes. The effects of NHERF1 on the distribution, mobility, signaling and internalization of apical and basolateral receptors will be studied. We predict that NHERF1 will anchor PTH1R to the apical actin cytoskeleton causing a decrease in the lateral mobility and rate of internalization. Furthermore, this tethering is expected to lead to ligand-induced calcium transients. Disruption of anchoring with NHERF1 knockdowns, or expression of receptor constructs unable to bind NHERF1 or NHERF1 mutants that do not bind the cytoskeleton are expected to reverse the polarizing effects of NHERF1 and lead to uniform PTH1R behavior throughout the cell. These studies will be conducted using various live-cell imaging techniques such as fluorescence recover after photobleaching, quantum dot single molecule tracking, optical measurements of cAMP and calcium and image cross-correlation analysis. These studies will enhance our understanding of the regulation of PTH1R by NHERF1 in the physiologically relevant setting of the proximal convoluted tubule. Since disorders of PTH1R and NHERF1 in the proximal tubules lead to disruption of phosphate homeostasis, better understanding of their physiology may lead to new treatment strategies for phosphatemia and phosphoruria. Because NHERF1 interacts with a wide range of GPCRs, ion channels and tyrosine kinases, extensions of these studies could translate into treatments for numerous disorders.