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: 8426172
• 负责人: Jean-Pierre Vilardaga
• 金额: $ 30.36万
• 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/8426172
• 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 Profile; 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; 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.

描述（由申请人提供）：B家族的PTH/PTHRP受体（PTHR），G蛋白偶联受体（GPCR），传输甲状旁腺激素（PTH）和PTH相关肽（PTHRP）信号，以启动和调节骨骼和肾脏物理学中的重要生物化学过程。尚不清楚该单个受体如何区分两个配体信号系统：PTH；内分泌和稳态，以及PTHRP；发育和各种器官生物学的旁分泌介体。目前尚不清楚为什么在临床环境中PTH（1-34）会刺激比PTHRP（1-36）（1-36）的血清1,25-二羟基 - 维胺-D，钙和骨吸收标记的延长增加，当时是通过持续浸出的imimic和Mimic Paryry的杂物，而不是PTHRP（1-36）。恶性。现在，我们推进了一个全面的模型来考虑这些活动。我们最近的研究表明，PTH（1-34）通过诱导培养细胞中的延长营地反应而与PTHRP（1-36）区分开来，而在受体水平上介导的体内，而不是通过配体的扩展生物利用度介导的体内。我们发现，在营地生产的时间范围内，PTHRP（1-36）的作用仅限于细胞表面，而PTH（1-34）被运输到内部化的亚细胞隔室，在该隔室中它与PTHR形成稳定的复合物，并继续刺激营地产量。这种明显的差异提供了一种机械基础，PTH和PTHRP引起了明显不同的反应，并表明PTHR信号向CAMP可以从细胞内域继续进行。基于这些新发现的结果，我们提出了一个中心假设，即PTHR的cAMP生产既发生在质膜和细胞内结构域，却有不同的寿命对细胞信号传导产生不同的后果。这个概念在此处描述的最新发现和初步数据的支持下，挑战了GPCR触发的CAMP生产的经典范式，仅源自细胞膜。 拟议的实验旨在确定a）PTHR触发的持续营地反应的机制； b）持续cAMP水平在细胞信号传导中的后果。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天然环境中进行实验测试。这些实验将有助于对活细胞本机环境中PTH/PTHRP/PTHR系统的分子和运输机制的基本了解，这是指导骨骼和矿物质疾病的更安全，更有效的药物的发展。