Internalized PTH receptor: New Consequence for Cell Signaling

描述（由申请人提供）：PTH/PTHrP受体（PTHR）是B家族的G蛋白偶联受体（GPCR），传递甲状旁腺激素（PTH）和PTH相关肽（PTHrP）信号，启动和调节骨和肾脏生理中的重要生化过程。目前尚不清楚这种单一受体如何区分两种配体信号系统：PTH；内分泌和体内平衡，以及PTHrP；发育和多种器官生物学的旁分泌介质。当配体通过持续输注来模拟原发性甲状旁腺功能亢进和恶性肿瘤的体液性高钙血症时，为什么在临床环境中，PTH（1-34）比PTHrP（1-36）更能刺激血清1,25-二羟基维生素d、钙和骨吸收标志物水平的延长。我们现在提出一个综合模型来解释这些活动。我们最近的研究表明，PTH（1-34）与PTHrP（1-36）的区别是通过在培养细胞和体内诱导长时间的cAMP反应，这是在受体水平上介导的，而不是通过延长配体的生物利用度。我们发现，在cAMP产生的时间框架内，PTHrP（1-36）的作用仅限于细胞表面，而PTH（1-34）被运输到内化的亚细胞区室，在那里它与PTHR形成稳定的复合物，并继续刺激cAMP的产生。这种显著的差异提供了PTH和PTHrP诱导明显不同反应的机制基础，并表明PTHR向cAMP的信号传导可以从细胞内域继续进行。基于这些新发现，我们提出了一个中心假设，即PTHR产生的cAMP既发生在质膜上，也发生在细胞内，具有不同的生命周期，对细胞信号传导有不同的影响。我们最近的研究结果和本文描述的初步数据支持了这一概念，挑战了由gpcr触发的cAMP产生仅起源于细胞膜的经典范式。拟议的实验旨在确定a) PTHR触发持续cAMP反应的机制；b)持续cAMP水平对细胞信号传导的影响。这些将通过光学技术以及药理学、生物化学和蛋白质组学方法在活细胞的天然环境中进行实验测试。这些实验将有助于对活细胞天然环境中PTH/PTHrP/PTHR系统激活和信号转导的分子和运输机制有一个基本的认识，这对指导开发更安全、更特异、更有效的骨和矿物质疾病药物是必要的。

DESCRIPTION (provided by applicant): The PTH/PTHrP receptor (PTHR), a G protein-coupled receptor (GPCR) of the B family, transmits both parathyroid hormone (PTH) and PTH-related Peptide (PTHrP) signals to initiate and regulate vital biochemical processes in bone and renal physiology. It is unknown how this single receptor discriminates between the two ligand signaling systems: PTH; endocrine and homeostatic, and PTHrP; a paracrine mediator of developmental and diverse organ biology. It is unclear also why in a clinical setting PTH(1-34) stimulates more prolonged increases in serum levels of 1,25-dihydroxy-vitamin-D, calcium, and bone resorption markers than does PTHrP(1-36), when the ligands are administered by continuous infusion so as to mimic conditions of primary hyperparathyroidism and humoral hypercalcemia of malignancy. We now advance a comprehensive model to account for these activities. Our recent studies show that PTH(1-34) differentiates itself from PTHrP(1-36) by inducing prolonged cAMP responses in cultured cells, and in vivo, which are mediated at the receptor level, and not by extended bioavailability of ligands. We discovered that during the time frame of cAMP production, PTHrP(1-36) action, is restricted to the cell surface, whereas PTH(1-34) trafficked to internalized sub-cellular compartments where it forms a stable complex with the PTHR, and continues to stimulate cAMP production. Such marked differences provide a mechanistic basis whereby PTH and PTHrP induce distinctly different responses and suggests that PTHR signaling to cAMP can continue from intracellular domains. Based on these novel findings, we propose the central hypothesis that cAMP production by the PTHR occurs both at the plasma membrane and from intracellular domains, with distinct lifetimes that have different consequences for cell signaling. This concept, supported by our recent findings and preliminary data described here, challenge the classical paradigm that cAMP production triggered by GPCRs originates exclusively at the cell membrane. The proposed experiments seek to determine a) the mechanisms of sustained cAMP responses triggered by the PTHR; and b) the consequences of sustained cAMP levels for cell signaling. These will be experimentally tested in the native environment of living cells by using optical technologies, as well as pharmacological, biochemical and proteomic approaches. These experiments will contribute to a fundamental understanding of the molecular and trafficking mechanisms of activation and signaling of the PTH/PTHrP/PTHR system in the native environment of living cells, which are needed to guide the development of safer and more specific and effective drugs for bone and mineral diseases. PUBLIC HEALTH RELEVANCE: The goal of this research plan is to understand how the medically important parathyroid hormone receptor transmits both parathyroid hormone (PTH) and PTH-related peptide (PTHrP) signals to regulate different physiological processes: PTH, endocrine and homeostatic regulates concentrations of calcium, phosphate ions, and vitamin D in blood and extracellular fluids, and PTHrP, a paracrine mediator of developing tissues such as bone. Pharmacological, biochemical and biophysical methods will be employed to advance a comprehensive model at the molecular and cellular levels to account for the functional differences between PTH and PTHrP, which will guide new therapies for osteoporosis.