Internalized PTH receptor: New Consequence for Cell Signaling

描述(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)和甲状旁腺激素相关肽(PTHrP)信号，启动和调节骨骼和肾脏生理中的重要生化过程。目前还不清楚这个单一的受体如何区分两个配体信号系统：PTH；内分泌和稳态，以及PTHrP；发育和不同器官生物学的旁分泌介体。也不清楚为什么在临床环境中，当通过持续输注配体来模拟原发性甲状旁腺功能亢进症和恶性肿瘤体液高钙血症时，PTH(1-34)比PTHrP(1-36)刺激血清1，25-二羟基维生素D、钙和骨吸收标记物水平的持续增加的时间更长。我们现在提出一个全面的模型来解释这些活动。我们最近的研究表明，PTH(1-34)通过在培养细胞和体内诱导长时间的cAMP反应而区别于PTHrP(1-36)，这种反应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来实现的。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)则被运输到内化的亚细胞室，在那里它与PTHR形成稳定的复合体，并继续刺激cAMP的产生。这些显著的差异提供了PTH和PTHrP引起明显不同反应的机制基础，并表明PTHR对cAMP的信号可以从细胞内域继续。基于这些新的发现，我们提出了中心假设，即PTHR产生cAMP既发生在质膜上，也发生在细胞内区域，具有不同的寿命，对细胞信号有不同的影响。这一概念得到了我们最近的发现和这里描述的初步数据的支持，挑战了GPCRs引发的cAMP产生仅起源于细胞膜的经典范式。拟议的实验试图确定a)PTHR触发持续的cAMP反应的机制；以及b)持续的cAMP水平对细胞信号的影响。这些将通过使用光学技术以及药理学、生化和蛋白质组学方法在活细胞的自然环境中进行实验测试。这些实验将有助于从根本上了解活细胞自然环境中PTH/PTHrP/PTHR系统的激活和信号传递的分子和运输机制，这是指导开发更安全、更特异和有效的骨和矿物质疾病药物所必需的。 公共卫生相关性：这项研究计划的目标是了解具有重要医学意义的甲状旁腺激素受体如何传递甲状旁腺激素(PTH)和甲状旁腺激素相关肽(PTHrP)信号来调节不同的生理过程：PTH、内分泌和稳态调节血液和细胞外液中钙、磷和维生素D的浓度，以及甲状旁腺激素(PTHrP)，它是发育组织(如骨骼)的旁分泌介质。将利用药理学、生物化学和生物物理学的方法，在分子和细胞水平上提出一个全面的模型，以解释PTH和PTHrP之间的功能差异，这将指导骨质疏松的新疗法。

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.