DUAL SIGNAL TRANSDUCTION VIA THE PTH RECEPTOR

本研究提案的总体目标是建立 启动的双重信号转导的功能意义 骨细胞中甲状旁腺激素受体（PTHR）的占据。 一 单一类型的克隆PTHR可以与cAMP和肌醇脂质偶联 途径。 虽然PTH的许多生理作用已被证明 仅或主要由cAMP介导， 磷酸肌醇衍生的第二信使尚不清楚，尤其是在骨中 细胞 尽管如此，我们和其他人已经确定了某些 PTH在成骨细胞中的cAMP非依赖性作用。 调解人 这些不依赖cAMP的作用是未知的。 在新的实验中 提出的两个假设将被测试：1。某些特定的生理 PTH对人成骨细胞的作用不依赖于蛋白质 激酶A激活，是由肌醇脂质的产物介导的 路径; 2。PTHR不与磷酸肌醇偶联， 某些人成骨细胞样细胞中的通路（例如，SaOS-2细胞），可以 在生物化学上和功能上与这个转导系统结合， 适当的异源系统，证明特定的细胞 受体远端的因子在信号转导中起关键作用 通过PTHR。 提出了两个具体目标。1.机理研究 成骨细胞胞浆Ca 2+稳态。 调查将 重点研究：cAMP与肌醇脂质之间的反馈调节 通路;特异性G蛋白; PTH调节的磷脂酶C; 细胞内Ca 2+螯合和释放区室;和 PTHR在GH4 C1大鼠垂体细胞中的异源表达， 非洲爪蟾卵母细胞。2.肌醇脂质作用的功能研究 成骨细胞中的信号转导通路。 这些实验旨在 确定成骨细胞对PTH的某些生理反应是否 是或可以通过磷酸肌醇水解产物调节。 提出的实验结果将回答两个尖锐的重点 问题：1.为什么PTHR，它可以耦合到两个转导， 途径，在成骨细胞中通常不这样做;和2.存在一定 甲状旁腺素对成骨细胞的重要生理作用 肌醇脂质途径？因为PTH具有分解代谢和合成代谢 行为，了解调节这些对立效应的途径 将为开发新的基于机制的方法提供机会， 控制过度的骨骼软骨或缺乏骨形成， 重要的临床疾病，如骨质疏松症、甲状旁腺功能亢进症和 一些恶性肿瘤引起的高钙血症

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.