PTH1R REGULATION IN BONE BIOLOGY USING A MOUSE MODEL

描述(由申请人提供)：甲状旁腺激素(PTH)/甲状旁腺激素相关肽(PTHrP)受体(PTH/PTHrP受体或PTH1R)被其同源配体PTH或PTHrP激活，启动两个平行的过程：1)刺激细胞内的第二信使，导致对矿物质离子稳态和骨细胞发育、分化和成熟的生物效应；以及2)PTH1R的磷酸化，受体-配体复合体的内在化和由该受体介导的生物反应的脱敏。PTH1R的磷酸化、内化和脱敏的生理作用尚不清楚。我们最近定位了PTH1R上的磷酸化位点，并开发了一种磷酸化缺陷(PD)PTH1R，它在配体刺激的内化过程中存在缺陷。利用同源重组技术，我们在ES细胞系中用突变的pdPTH1R取代了正常的PTH1R，并已经培育出纯合子敲入(PD/PD)小鼠。帕金森病/帕金森病小鼠具有生育能力、存活能力和正常的血钙水平。然而，这些小鼠的甲状旁腺素水平和磷酸盐水平都很低。此外，这些小鼠对甲状旁腺素注射表现出延长和夸大的cAMP反应。甲状旁腺激素生物学中一个有趣的谜题是理解间歇性甲状旁腺素是如何促进骨形成的，而持续甲状旁腺素是如何促进骨吸收的。在特定目的1中，我们建议建立一种表达pdPTH1R的敲打小鼠模型，以了解PTH1R的磷酸化、内化和脱敏在骨发育、分化和成熟中的作用，以及体内间歇性给予PTH的合成代谢效应。在特定的目标2中，我们将研究pdPTH1R敲击小鼠的骨和软骨表型，以评估PTH1R的磷酸化和内化对骨和软骨细胞的发育、分化和成熟的调节至关重要的假说。在具体目标3中，我们将使用这个模型来检验这样的假设，即PTH1R的磷酸化和内化在PTH间歇给药的合成代谢作用的表达中起着重要作用。该项目目标的实现将增加我们对PTH/PTHrP受体的磷酸化和内在化如何影响PTH在骨骼中的表达(形成和吸收)的理解；这对于了解骨骼疾病和设计新的靶向预防和逆转这些疾病的治疗药物是必不可少的。

DESCRIPTION (provided by applicant): Activation of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (the PTH/PTHrP receptor or PTH1R) by its cognate ligands, PTH or PTHrP, initiates two parallel processes: 1) stimulation of intracellular second messengers leading to biologic effects on mineral ion homeostasis and bone cell development, differentiation, and maturation; and 2) phosphorylation of PTH1R, internalization of the receptor - ligand complexes and desensitization of the biologic responses mediated by this receptor. The physiological role of PTH1R phosphorylation, internalization and desensitization is not known. We have recently mapped the phosphorylation sites on PTH1R and developed a phosphorylation-deficient (pd) PTH1R, which is defective in ligand-stimulated internalization. Using homologous recombination techniques we have "knocked in" the mutant pdPTH1R to replace the normal PTH1R in ES cell lines and have already developed homozygous knock-in (pd/pd) mice. The pd/pd mice are fertile, viable, and normocalcemic. However, these mice have low PTH and low phosphate levels. In addition, these mice showed prolonged and exaggerated cAMP response to PTH injection. One intriguing puzzle in PTH biology is to understand how intermittent PTH administration promotes bone formation whereas continuous PTH administration promotes bone resorption. In Specific Aim 1 we propose to develop a knock in mouse model expressing the pdPTH1R to understand the role of PTH1R phosphorylation, internalization and desensitization in bone development, differentiation and maturation and in the anabolic effects of intermittent PTH administration in vivo. In Specific Aim 2 we shall examine the bone and cartilage phenotype of the pdPTH1R knock in mouse to assess the hypothesis that phosphorylation and internalization of the PTH1R is important for the regulation of bone and chondrocytic cell development, differentiation and maturation. In Specific Aim 3 we shall use this model to examine the hypothesis that PTH1R phosphorylation and internalization play an important role for the expression of the anabolic actions of intermittent PTH administration. Achievement of the goals of this project will increase our understanding of how phosphorylation and internalization of the PTH/PTHrP receptor affects the expression of PTH actions in bone (formation and resorption); this is essential to understand bone diseases and design new therapeutic agents targeted for their prevention and reversal.