PARATHYROID HORMONE RECEPTORS IN KIDNEY AND BONE

最近的证据表明，一个单一的G蛋白偶联受体， 负责甲状旁腺激素（PTH）的主要生物学效应 和PTH相关蛋白（PTHrP）。通过这种受体，这些 肽发挥重要的生理作用-甲状旁腺素作为一种主要的内分泌 血浆钙和骨骼稳态的调节剂，PTHrP作为 正常软骨内骨生长分化因子 发展此外，当适当施用时，PTH产生 对骨骼的合成代谢作用，并可能具有治疗价值， 骨质疏松症患者。因此，重要的是 详细了解PTH/PTHrP受体如何传递 将指导性信号导入目标细胞，以及信号传输是如何 监管. 以前的研究表明，信号转导， PTH/PTHrP受体涉及两个独立途径的激活- 腺苷酸环化酶/cAMP和磷脂酶C/Ca2+。这些途径 暴露于以下物质后对PTH或PTHrP失去反应性 急性激动剂（导致受体脱敏）或 慢性（导致受体下调）。这两个过程都是 被认为是靶细胞反应性的重要决定因素，但 我们对它们的分子基础几乎一无所知。最近的研究 证明PTH/PTHrR受体的大胞质尾区 包含受体表达、内吞作用和 磷酸化，这表明在调节 受体功能受体调节的机制将通过以下定义： 1)确定PTH/PTHrP受体的胞质尾区的作用 在受体磷酸化和同源脱敏中的作用。表达 人胚肾中的重组突变PTH/PTH rP受体293 细胞将被用作一个模型系统，以绘制尾部的站点， PTH刺激的磷酸化和脱敏所需; 2） 评估G蛋白偶联受体激酶（GRKs）在 PTH/PTHrP受体磷酸化和脱敏。一个知道的角色 GRK（β-肾上腺素能受体激酶，β-ARK）将由 PTH应答UMR中β-ARK显性抑制形式的表达 106成骨细胞骨肉瘤细胞，和潜在的新GRKs在这些 将通过逆转录酶-聚合酶链反应检查细胞 （RT-PCR）; 3）评估C末端尾在PTH/PTHrP中的作用 受体内吞和再循环。表达/诱变将用于 鉴定调节受体运输的特定序列，以及 确定它们在激动剂刺激的受体下调中的作用;以及 4)定义了极化表达的分子基础， PTH/PTHrP受体的表达和定位，在Madin- 达比犬肾（MDCK）上皮细胞。成功完成 这些研究将提供关于生物学 PTH和PTHrP的作用在其共同的水平上受到调节， 受体的

Recent evidence indicates that a single G-protein-coupled receptor is responsible for the major biological effects of parathyroid hormone (PTH) and PTH-related protein (PTHrP). Acting through this receptor, these peptides play essential physiological roles- PTH as a major endocrine regulator of plasma calcium and skeletal homeostasis, and PTHrP as a growth and differentiation factor required for normal endochondral bone development. Moreover, when appropriately administered, PTH produces anabolic effects on the skeleton and may have therapeutic value in patients with osteopenic disorders. For these reasons, it is important to gain a detailed understanding of how the PTH/PTHrP receptor transmits instructive signals into target cells, and how signal transmission is regulated. Previous studies have demonstrated that signal transduction by the PTH/PTHrP receptor involves activation of two independent pathways- adenylyl cyclase/cAMP and phospholipase C/Ca-i2+. These pathways are subject to loss of responsiveness to PTH or PTHrP following exposure to agonists either acutely (resulting in receptor desensitization) or chronically (resulting in receptor down-regulation). Both processes are thought to be important determinants of target cell responsiveness, but almost nothing is known about their molecular bases. Recent studies demonstrate that the large cytoplasmic tail of the PTH/PTHrR receptor contains important determinants of receptor expression, endocytosis, and phosphorylation, suggesting an important role in the regulation of receptor function. Mechanisms of receptor regulation will be defined by: 1) determining the role of the cytoplasmic tail of the PTH/PTHrP receptor in receptor phosphorylation and homologous desensitization. Expression of recombinant, mutated PTH/PTH rP receptors in human embryonic kidney 293 cells will be used as a model system to map the sites in the tail that are required for PTH-stimulated phosphorylation and desensitization; 2) evaluating the role of G-protein-coupled receptor kinases (GRKs) in PTH/PTHrP receptor phosphorylation and desensitization. The role of a know GRK (beta-adrenergic receptor kinase, beta-ARK) will be tested by expression of a dominant inhibitory form of beta-ARK in PTH-responsive UMR 106 osteoblastic osteosarcoma cells, and potentially novel GRKs in these cells will be examined by reverse transcriptase-polymerase chain reaction (RT-PCR); 3) assessing the role of the C-terminal tail in PTH/PTHrP receptor endocytosis and recycling. Expression/mutagenesis will be used to identify specific sequences that regulate receptor trafficking, and to determine their role in agonist-stimulated receptor down-regulation; and 4) defining the molecular basis of the polarized expression of the PTH/PTHrP receptor by expressing and localizing the receptor in Madin- Darby canine kidney (MDCK) epithelial cells. Successful completion of these studies will provide mechanistic insights into how the biological effects of PTH and PTHrP are regulated at the level of their common receptor.