Sustained cAMP signals triggered by internalized PTH receptor: new consequences f

由内化 PTH 受体触发的持续 cAMP 信号：新的后果

• 批准号: 8062026
• 负责人: Jean-Pierre Vilardaga
• 金额: $ 31.46万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8062026
• 关键词: Accounting; Arrestins; Biochemical; Biochemical Process; Biological Availability; Biology; Blood; Bone Resorption; Calcium; Cell membrane; Cell surface; Cells; Clinical; Complex; Continuous Infusion; Cultured Cells; Cyclic AMP; Data; Development; Disease; Endocrine; Endosomes; Environment; Extracellular Fluid; Family; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Goals; Hormones; Hypercalcemia; Hypercalcemia of Malignancy; Hyperparathyroidism; Intracellular Membranes; Ions; Kidney; Lead; Life; Ligands; Link; Mediating; Mediator of activation protein; Methods; Minerals; Modeling; Molecular; Optics; Organ; Osteoporosis; Parathyroid Hormone Receptor; Parathyroid gland; Pattern; Pharmaceutical Preparations; Physiological Processes; Physiology; Plasma; Production; Proteins; Proteomics; Receptor Signaling; Research; Serum; Signal Transduction; System; Technology; Testing; Time; Tissues; Vitamin D; base; bone; calcium phosphate; design; hormone regulation; in vivo; novel; paracrine; parathyroid hormone (1-34); parathyroid hormone-related protein; protein complex; public health relevance; receptor; research study; response; trafficking

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.

描述（由申请人提供）：PTH/PTHrP受体（PTHR）是B家族的一种G蛋白偶联受体（GPCR），可传递甲状旁腺激素（PTH）和PTH相关肽（PTHrP）信号，以启动和调节骨和肾生理学中的重要生化过程。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;内分泌和稳态，以及PTHrP;发育和多种器官生物学的旁分泌介体。当通过连续输注给予配体以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，在临床环境中，PTH（1-34）为什么比PTHrP（1-36）刺激血清中1，25-二羟基维生素D、钙和骨吸收标记物水平更长时间的增加也不清楚。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）通过诱导培养细胞中和体内的延长的cAMP应答而将其自身与PTHrP（1-36）区分开，所述延长的cAMP应答在受体水平介导，而不是通过延长配体的生物利用度。我们发现，在cAMP产生的时间范围内，PTHrP（1-36）作用限于细胞表面，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产生。这种显著的差异提供了一个机制的基础，PTH和PTHrP诱导明显不同的反应，并表明PTHR信号转导cAMP可以继续从细胞内结构域。基于这些新的发现，我们提出了一个中心假设，即由PTHR产生的cAMP发生在质膜和细胞内结构域，具有不同的寿命，对细胞信号传导具有不同的后果。这一概念得到了我们最近的发现和本文描述的初步数据的支持，挑战了经典的范式，即由GPCR触发的cAMP产生仅起源于细胞膜。 所提出的实验试图确定a）由PTHR触发的持续cAMP应答的机制;和B）持续cAMP水平对细胞信号传导的后果。这些将通过使用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。这些实验将有助于从根本上了解PTH/PTHrP/PTHR系统在活细胞的天然环境中的活化和信号传导的分子和运输机制，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。 公共卫生关系：本研究计划的目标是了解医学上重要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，以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，磷酸根离子和维生素D的浓度，以及PTHrP，发育组织（如骨骼）的旁分泌介质。将采用药理学、生物化学和生物物理学方法，在分子和细胞水平上推进一个综合模型，以解释PTH和PTHrP之间的功能差异，这将指导骨质疏松症的新疗法。