EBP50 REGULATION OF PTH RECEPTOR IN BONE AND KIDNEY

描述(申请人提供)：该项目的长期目标是阐明甲状旁腺激素受体(PTH1R)作用的细胞机制。PTH1R通过对肾脏和骨骼的作用来调节细胞外钙和磷的稳态。与其他G蛋白偶联受体一样，PTH1R表现出一个激活、脱敏、内化和再增敏的循环过程。受体脱敏提供了一种保护细胞免受过度刺激的机制，而再增敏则保护细胞免受长期不活动和激素抵抗。然而，与大多数其他受体不同的是，PTH1R在其激活过程中表现出相当大的细胞和组织特异性差异。这些差异不能归因于选择性剪接受体形式、受体丰度或G蛋白的可获得性。最近的证据表明，胞质适配器蛋白Ezrin结合蛋白50 kD(EBP50)可能参与细胞特异性PTH1R信号转导和内化。拟议研究的中心目标是研究EBP50在PTH1R循环的各个方面的相互作用和调节活性。我们开发了四个特定的目标来验证EBP50调节PTH1R的配体特异性反应的统一假设。目的1研究EBP50对细胞特异性PTH1R激活的影响。目的2将描述EBP50在PTH1R脱敏中的作用。目标3将确定参与PTH1R内化的EBP50的结构决定因素。目的4将确定EBP50在PTH1R循环中的作用。计划中的研究采用了一系列细胞生物学、生化和分子生物学技术，这些技术将应用于作为甲状旁腺素作用主要靶点的特定肾脏和骨细胞。初步数据为许多拟议工作提供了临时支持并确定了其可行性。计划中的研究将提供关于PTH1R调节细胞外钙稳态的机制和作用的新的和重要的信息。这些结果将为更好地理解PTH1R作用的启动和终止提供依据。这些结果可能提示导致甲状旁腺激素抵抗的其他病理生理机制，并导致新的治疗机会。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the cellular mechanisms of parathyroid hormone receptor (PTH1R) action. The PTH1R regulates extracellular calcium and phosphate homeostasis by its actions on kidney and bone. Like other G protein-coupled receptors, the PTH1R exhibits a cyclical process of activation, desensitization, internalization, and resensitization. Receptor desensitization provides a mechanism to protect cells against excessive stimulation, while resensitization guards cells against prolonged inactivity and hormone resistance. Unlike most other receptors, however, the PTH1R exhibits considerable cell- and tissue-specific differences in its activation. These differences cannot be ascribed to alternatively spliced receptor forms, receptor abundance, or G protein availability. Recent evidence suggests that the cytoplasmic adaptor protein ezrin-binding protein 50 kD (EBP50) may contribute to cell-specific PTH1R signaling and internalization. The central goal of the proposed studies is to examine the interaction and modulatory activity of EBP50 on all aspects of PTH1R cycling. Four specific aims are developed to test the unifying hypothesis that EBP50 regulates ligand-specific responses of the PTH1R. Aim 1 will characterize the effects of EBP50 on cell-specific PTH1R activation. Aim 2 will describe EBP50 effects on PTH1R desensitization. Aim 3 will identify structural determinants of EBP50 that are involved in PTH1R internalization. Aim 4 will determine the effects of EBP50 on PTH1R recycling. The planned studies employ an array of cell biological, biochemical, and molecular biological techniques that will be applied to specific kidney and bone cells that are the primary targets of PTH action. Preliminary data provide provisional support and establish the feasibility for much of the proposed work. The planned studies will yield novel and important information on the mechanism and role by which the PTH1R regulates extracellular calcium homeostasis. The results will provide greater understanding of the initiation and termination of PTH1R action. The outcomes may suggest additional pathophysiological mechanisms causing PTH resistance and lead to new treatment opportunities.