DUAL SIGNAL TRANSDUCTION VIA THE PTH RECEPTOR

这项研究建议的总体目标是建立 启动双重信号转导的功能意义 甲状旁腺激素受体(PTHR)在骨细胞中的占有率。一个 单型克隆的PTHR可与cAMP和肌醇脂质偶联 小路。虽然甲状旁腺激素的许多生理作用已被证明 完全或主要由cAMP调节，其功能作用 肌醇磷脂衍生的第二信使尚不清楚，尤其是在骨骼中。 细胞。然而，我们和其他人已经确定了某些 甲状旁腺素在成骨细胞中的cAMP非依赖性作用。的调解人 这些独立于阵营的行动尚不为人所知。在新的实验中 提出的两个假说将被检验：1.某些特定的生理 甲状旁腺素对人成骨细胞的作用不依赖于蛋白质 由肌醇脂类产物介导的激酶A激活 2.不与肌醇磷脂偶联的PTHR 在某些人成骨样细胞(例如，SAOS-2细胞)中的通路，可以 在生物化学和功能上偶联到这个转导系统 合适的异源系统，证明了特定的细胞 受体末端的因子在信号转导中起着关键作用。 通过PTHR。提出了两个具体目标。1.隧道开挖的机理研究 成骨细胞内钙离子的动态平衡。调查将会 焦点：cAMP和肌醇脂质之间的反馈调节 途径；特异性G蛋白；甲状旁腺素调节的磷脂酶C； 细胞内钙离子封存和释放隔间；以及 甲状旁腺素受体在GH4C1大鼠垂体细胞中的异源表达 非洲爪哇卵母细胞。2.肌醇脂类作用的功能研究 成骨细胞中的转导途径。这些实验旨在 确定成骨细胞对甲状旁腺素的某些生理反应 是或可以通过磷脂酰肌醇的水解产物来调节。 提出的实验结果将回答两个尖锐的焦点 问：1.为什么可以连接两个转导的PTHR 在成骨细胞中，通常不会有这样的通路；以及2.确定 甲状旁腺素在成骨细胞中的重要生理作用 肌醇脂质途径？因为甲状旁腺素既有分解代谢又有合成代谢 行为，了解调节这些相反效果的途径 将提供机会来开发新的基于机制的方法 控制过度的骨骼分解代谢或缺乏骨形成 重要的临床疾病，如骨质疏松症、甲状旁腺功能亢进症和 一些恶性的高钙血症。

The overall objective of this research proposal is to establish the functional significance of dual signal transduction initiated by occupancy of the parathyroid hormone receptor (PTHR) in bone cells. A single type of cloned PTHR can couple to both the cAMP and inositol lipid pathways. While many of the physiological actions of PTH have been shown to be mediated solely or primarily by cAMP, the functional role of phosphoinositide-derived second messengers is unclear, especially in bone cells. Nevertheless, we and others have identified certain cAMP-independent actions of PTH in osteoblastic cells. The mediators of these cAMP-independent actions are not known. In the new experiments proposed two hypotheses will be tested: 1. certain specific physiological actions of PTH on human osteoblasts, which are independent of protein kinase A activation, are mediated by products of the inositol lipid pathway; and 2. the PTHR, which does not couple to the phosphoinositide pathway in certain human osteoblast-like cells (e.g., SaOS-2 cells), can couple biochemically and functionally to this transduction system in appropriate heterologous systems, demonstrating that specific cellular factors distal to the receptor play crucial roles in signal transduction via the PTHR. Two specific aims are proposed. 1. Mechanistic studies on cytosolic Ca2+ homeostasis in osteoblastic cells. Investigations will focus on: feedback regulation between the cAMP and inositol lipid pathways; specific G proteins; PTH-regulated phospholipase C; intracellular Ca2+ sequestration and release compartments; and heterologous expression of the PTHR in GH4C1 rat pituitary cells and Xenopus oocytes. 2. Functional studies on the role of the inositol lipid transduction pathway in osteoblasts. These experiments are designed to determine whether certain physiological responses to PTH in osteoblasts are or can be regulated via products of phosphoinositide hydrolysis. Results of the experiments proposed will answer two sharply focused questions: 1. why does the PTHR, which can couple to two transduction pathways, frequently not do so in osteoblasts; and 2. are certain important physiological actions of PTH on osteoblasts mediated by the inositol lipid pathway? Because PTH has both catabolic and anabolic actions, understanding the pathways regulating these opposing effects will offer the opportunity to develop new mechanism-based approaches to control excessive skeletal catabolism or deficient bone formation in such important clinical disorders as osteoporosis, hyperparathyroidism, and in some of the hypercalcemias of malignancy.