PTH1R REGULATION IN BONE BIOLOGY USING A MOUSE MODEL

描述（申请人提供）：甲状旁腺激素（PTH）/PTH相关肽（PTHrP）受体的激活（PTH/PTHrP受体或PTH 1 R）通过其同源配体PTH或PTHrP启动两个平行过程：1）刺激细胞内第二信使，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;和2）PTH 1 R的磷酸化、受体-配体复合物的内化和由该受体介导的生物反应的脱敏。PTH 1 R磷酸化、内化和脱敏的生理作用尚不清楚。我们最近绘制了PTH 1 R上的磷酸化位点，并开发了磷酸化缺陷型（PD）PTH 1 R，它在配体刺激的内化中有缺陷。使用同源重组技术，我们已经“敲入”突变型pdPTH 1 R，以取代ES细胞系中的正常PTH 1 R，并已开发出纯合敲入（PD/PD）小鼠。PD/PD小鼠具有生育能力、存活能力和正常血钙。然而，这些小鼠具有低PTH和低磷酸盐水平。此外，这些小鼠对PTH注射表现出延长和夸大的cAMP反应。PTH生物学中一个有趣的难题是了解间歇性PTH给药如何促进骨形成，而连续性PTH给药如何促进骨吸收。在具体目标1中，我们建议开发一种表达pdPTH 1 R的敲入小鼠模型，以了解PTH 1 R磷酸化、内化和脱敏在骨发育、分化和成熟中的作用，以及间歇性PTH给药在体内的合成代谢作用。在具体目标2中，我们将检查pdPTH 1 R敲除小鼠的骨和软骨表型，以评估PTH 1 R的磷酸化和内化对于骨和软骨细胞发育、分化和成熟的调节很重要的假设。在具体目标3中，我们将使用该模型来检验PTH 1 R磷酸化和内化对间歇性PTH给药的合成代谢作用的表达起重要作用的假设。该项目目标的实现将增加我们对PTH/PTHrP受体的磷酸化和内化如何影响骨中PTH作用（形成和吸收）的表达的理解;这对于理解骨疾病和设计针对其预防和逆转的新治疗药物至关重要。

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.