Regulation of the PTH/PTHrP Receptor

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）刺激细胞内第二信使，导致矿物离子稳态和骨细胞发育、分化和成熟的生物学效应；2) PTH1R的磷酸化，受体-配体复合物的内化以及由该受体介导的生物反应的脱敏。PTH1R磷酸化、内化和脱敏的生理作用尚不清楚。我们最近绘制了PTH1R的磷酸化位点，并发现了一种磷酸化缺陷（pd） PTH1R，它在配体刺激的内化中存在缺陷。利用同源重组技术，我们“敲入”突变的pdPTH1R来取代正常的PTH1R。我们将使用pdPTH1R敲入小鼠模型来了解PTH1R磷酸化、内化和脱敏在不同生理条件下钙稳态中的作用（Specific Aim 1）。我们还将使用体外细胞系模型来解剖参与PTH1R磷酸化和内化的分子（Specific Aim 2）。三种天然存在的组成活性PTH1R突变体为研究受体的分子机制提供了独特的机会

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. We shall use the pdPTH1R knock in mouse model to understand the role of PTH1R phosphorylation, internalization and desensitization in calcium homeostasis in different physiological condition (Specific Aim 1). We shall also use in vitro cell line models to dissect the molecules involved in PTH1R phosphorylation and internalization (Specific Aim 2). Three naturally-occurring constitutively-active PTH1R mutants provide a unique opportunity to study the molecular mechanism of receptor regulation. The difference in their basal activity and agonist simulation may reflect intrinsic differences in phosphorylation and internalization. Understanding how these mutants are regulated will shed light into the molecular mechanisms of receptor phosphorylation, internalization and desensitization (Specific Aim 3). PTH analogs with unique binding and signaling properties, developed in Project I, will be tested for selective effects on internalization and desensitization. Identification of analogs which differentiate receptor activation from receptor internalization will provide a unique tool to understand the molecular mechanisms of receptor internalization and desensitization (Specific Aim 4).