PARATHYROID HORMONE RECEPTORS IN KIDNEY AND BONE

最近的证据表明，单一的G蛋白偶联受体是 甲状旁腺激素(PTH)的主要生物学效应 和甲状旁腺素相关蛋白(PTHrP)。通过这种受体发挥作用，这些 多肽发挥着重要的生理作用--甲状旁腺素作为一种主要的内分泌 血浆钙和骨骼动态平衡的调节剂，PTHrP作为一种 正常软骨内骨所需的生长和分化因子 发展。此外，如果给药得当，甲状旁腺素可产生 对骨骼的合成代谢作用，可能有治疗价值 患有骨质疏松症的患者。出于这些原因，重要的是 详细了解甲状旁腺素/甲状旁腺素受体如何传递 指令信号进入目标小区，以及信号如何传输 受监管的。先前的研究已经证明，信号转导通过 甲状旁腺素/甲状旁腺素受体涉及两个独立的通路的激活- 腺酰环化酶/cAMP和磷脂酶C/Ca-i2+。这些路径是 暴露于甲状旁腺素或甲状旁腺素rP后失去对甲状旁腺素或甲状旁腺素的反应性 激动剂要么剧烈(导致受体脱敏)，要么 慢性(导致受体下调)。这两个过程都是 被认为是目标细胞反应性的重要决定因素，但 对它们的分子碱基几乎一无所知。最新研究 证明甲状旁腺素/甲状旁腺素受体的大细胞质尾巴 包含重要的受体表达决定因素、内吞作用和 磷酸化，提示在调节 受体功能。受体调节机制的定义如下： 1)确定PTH/PTHrP受体胞质尾部的作用 受体磷酸化和同源脱敏。的表达 人胚胎肾脏中重组、突变的甲状旁腺素/甲状旁腺素受体293 细胞将被用作模型系统，以绘制尾部中的 甲状旁腺素刺激的磷酸化和脱敏所需；2) G蛋白偶联受体激酶(GRKs)在心肌梗死中的作用 甲状旁腺素/甲状旁腺素受体的磷酸化和脱敏。知情者的角色 GRK(β-肾上腺素能受体激酶，β-ARK)将通过 甲状旁腺素反应UMR中优势抑制形式β-Ark的表达 106成骨性骨肉瘤细胞和潜在的新的GRK 细胞将通过逆转录-聚合酶链式反应进行检测 (RT-PCR)；3)评估C端尾在甲状旁腺激素/甲状旁腺激素相关蛋白中的作用 受体内吞和循环。表达/诱变技术将用于 确定调节受体交易的特定序列，并 确定它们在激动剂刺激的受体下调中的作用； 4)定义极化表达的分子基础 甲状旁腺素/甲状旁腺素受体在MADIN中的表达和定位 Darby犬肾上皮细胞。成功完成 这些研究将提供对生物如何 甲状旁腺素和甲状旁腺素的作用在它们共同的水平上被调节 受体。

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.