Novel Regulation of PTH Receptor Functions in Bone

描述（由申请人提供）：1型甲状旁腺激素受体（PTH1R）是g蛋白偶联受体（gpcr）的一员，可介导甲状旁腺激素的作用以维持骨矿物质的稳态。成骨细胞功能障碍导致骨质流失被认为是骨质疏松症的关键机制。间歇给药PTH（1-34）刺激患者和实验动物的骨形成。持续输注PTH(1-34)，模拟临床甲状旁腺功能亢进的病理变化，引起骨吸收。终末期肾病患者出现全身性矿物质和骨代谢紊乱，如肾性骨营养不良。PTH1R下调和骨骼对PTH的抵抗不仅降低了PTH治疗骨质疏松症的疗效，而且还发生在继发性甲状旁腺功能亢进、肾性骨营养不良等疾病中。防止PTH1R下调是一种补充策略，可能对治疗这些疾病有用。虽然PTH受体的信号通路已经较为清楚，但其调控PTH1R功能的机制仍有待研究。突触后密度95/大盘/闭塞带（PDZ）支架蛋白包括一类关键的gpcr相互作用蛋白，可以强烈影响gpcr的信号传导和运输。通过新开发的不同PDZ结构域的蛋白质组学阵列和共免疫沉淀实验，我们鉴定了两个新的PTH1R相关蛋白。这些PDZ支架蛋白，MAGI-3和嗜脊髓蛋白，在成骨细胞中内源性表达。MAGI-3和嗜脊髓素均可增加pth诱导的成骨细胞cAMP的形成。基于这些观察，我们假设MAGI-3和嗜脊髓蛋白调节PTH1R的功能，并保护骨骼免受受体下调。为了验证这一假设，提出了三个具体目标。目的1将确定MAGI-3和嗜脊髓蛋白对成骨细胞PTH1R信号传导的影响。在Aim 2中，我们将确定MAGI-3和嗜脊髓蛋白是否能抑制成骨细胞中PTH1R的下调。目的3将确定嗜脊髓素对间歇性甲状旁腺激素治疗小鼠骨形成和骨吸收的作用。这些研究的成功完成将为骨中PTH1R信号的调控、转运和MAGI-3和嗜脊髓蛋白的功能提供新的重要信息。从这些研究中获得的知识将为骨质疏松症和其他与PTH1R下调相关的疾病（如继发性甲状旁腺功能亢进和肾性骨营养不良）的治疗提供重要的见解。

DESCRIPTION (provided by applicant): The type 1 parathyroid hormone receptor (PTH1R), a member of G-protein coupled receptors (GPCRs), mediates PTH actions to maintain bone mineral homeostasis. Osteoblast dysfunction leading to bone loss is thought to be a key mechanism in osteoporosis. Intermittent administration of PTH (1-34) stimulates bone formation in patients and in experimental animals. Continuous infusion of PTH (1-34), which mimics pathological changes in clinical hyperparathyroidism, causes bone resorption. Patient with end-stage renal disease develops a systemic disorder of mineral and bone metabolism, such as renal osteodystrophy. PTH1R down-regulation and skeletal resistance to PTH not only reduce the therapeutic effect of PTH treatment for osteoporosis, but also occur in diseases including secondary hyperparathyroidism and renal osteodystrophy. To protect against the PTH1R down-regulation is a complementary strategy that may be useful for treatment of these diseases. Although the signaling pathways of PTH receptor are reasonably well understood, the mechanism of regulation of PTH1R functions remains to be characterized. Postsynaptic density 95/discs large/zona occludens (PDZ) scaffolding proteins comprise a key class of GPCRs-interacting proteins that can strongly influence signaling and trafficking of GPCRs. We identified two novel PTH1R associating proteins by using a newly developed proteomic array of distinct PDZ domains and coimmunoprecipitation experiments. These PDZ scaffolding proteins, MAGI-3 and spinophilin, are endogenously expressed in osteoblasts. Both MAGI-3 and spinophilin increase PTH-induced cAMP formation in osteoblasts. Based on these observations, we hypothesize that MAGI-3 and spinophilin modulate PTH1R functions and protect against receptor down-regulation in bone. Three specific aims are developed to test this hypothesis. Aim 1 will determine the effects of MAGI-3 and spinophilin on PTH1R signaling in osteoblasts. In Aim 2, we will identify whether MAGI-3 and spinophilin protect against PTH1R down-regulation in osteoblasts. Aim 3 will define the role of spinophilin on bone formation bone resorption in mice treated with intermittent PTH. Successful completion of the proposed studies will provide novel and important information on the regulation of PTH1R signaling, trafficking, and functions by MAGI-3 and spinophilin in bone. The knowledge gained from these studies will provide important insight into therapeutics for the treatment of osteoporosis and other diseases related to PTH1R down-regulation such as secondary hyperparathyroidism and renal osteodystrophy.