EVOLUTION OF THE PTH/PTHRP RECEPTOR AND ITS LIGANDS

甲状旁腺激素（parathyroid hormone，PTH）是哺乳动物体内最重要的调节因子 钙代谢，由甲状旁腺合成和分泌 腺体;这些是缺席的鱼和第一次出现在无尾类之前或 在变形过程中。 PTH相关肽（PTHrP），其具有有限的N- 与PTH的末端氨基酸序列同源性，通常是导致 恶性肿瘤的体液性高钙血症（HHM）综合征。 其正常 生理意义知之甚少。 PTHrP的表达 在早期胚胎发生期间以及在多个胎儿和成人组织中， 然而，这表明自分泌或旁分泌功能， PTHrP和PTH共有的受体。 配体及其 因此，受体可能对人类具有相当重要意义 发展 进化研究作为一种评估 激素、生长因子及其效应途径的功能 已证明对各种内分泌/旁分泌系统有用，因此提供 几个独特的机会来评估PTH和PTHrP：1）隔离 用于配体/受体结构/功能分析的新型类似物 相互作用和功能重要受体的定义 域，2）新颖的，技术上更容易的工具来研究功能， 在哺乳动物系统中不同或可能不太明显，3）强大 系统的遗传分析，和4）模型系统，以研究 PTHrP（和PTH）对人类发育的重要性。 感悟 PTHrP的生理功能，以确定PTH是如何进化的 成为哺乳动物矿物质离子代谢的主要调节剂， 评估两种配体如何通过共同的 这是本书的第一个目标。 因此，我们建议 在目的I中，为了研究这两种配体的进化历史， 受体，或可能结合一种或另一种配体的受体 优选或专门通过分离编码这些蛋白质的cDNA 从鸡，非洲爪蟾，和鱼，并随后从 无脊椎动物 PTH、PTHrP的功能评价及其 在Aim II中，来自不同物种的共同受体将扩大我们的 配体之间的结构/功能关系的知识， 受体。 配体的独特的物种特异性特征 和/或受体将有助于进一步在结构上和 受体的功能重要结构域。 结果发现， 可以允许设计用于治疗 高钙血症由于高甲状旁腺激素或HHM综合征，并可能 对于理解和治疗 治疗骨质疏松症的方法 在目标III中，将使用上述工具 在变态前和变态后的无尾两栖类中鉴定产生PTH的细胞 在甲状旁腺首次出现的地方，在有甲状旁腺激素的鱼类中， 缺乏解剖学上定义的甲状旁腺。 此外，PTH和PTHrP 将在鱼体内进行评价，靶器官含有受体， 两种配体将通过原位北方印迹分析鉴定 杂交和免疫组织化学。 PTHrP（和PTH）在 在非洲爪蟾卵母细胞中评价早期胚胎发生， 卵裂球和整个胚胎，通过评估中胚层诱导， 模式化

Parathyroid hormone (PTH), the most important regulator of mammalian calcium metabolism, is synthesized and secreted by the parathyroid glands; these are absent in fish and first appear in anurans before or during metamorphosis. PTH-related peptide (PTHrP), which has limited N- terminal amino acid sequence homology with PTH, is frequently responsible for the humoral hypercalcemia of malignancy (HHM) syndrome. Its normal physiological significance is poorly understood. Expression of PTHrP during early embryogenesis and in multiple fetal and adult tissues, however, suggests auto- or paracrine functions which are mediated through a receptor that is shared by PTHrP and PTH. The ligands and their receptor are thus likely to be of considerable importance for human development. Evolutionary studies as an approach to assess the function(s) of hormones, growth factors, and their effector pathways have proven useful for various endocrine/paracrine systems, and thus offer several unique opportunities to evaluate PTH and PTHrP: 1) the isolation of novel analogs for the structure/function analysis of ligand/receptor interaction and the definition of functionally important receptor domains, 2) novel, technically easier tools to study functions that are different or perhaps less obvious in mammalian systems, 3) powerful systems for genetic analysis, and 4) model systems to study the importance of PTHrP (and PTH) for human development. To gain insights into the physiological functions of PTHrP, to establish how PTH evolved into the major regulator of mammalian mineral ion metabolism, and to evaluate how both ligands diversified their actions through a common receptor, are the first goals of this proposal. We, therefore, propose in Aim I to study the evolutionary history of both ligands, their shared receptor, or possibly receptors that bind one or the other ligand preferentially or exclusively by isolating cDNAs encoding these proteins from chicken, Xenopus laevis, and fish, and subsequently from invertebrate species. the functional evaluation of PTH, PTHrP, and their common receptor from various species will, in Aim II, expand our knowledge of the structure/function relationship between ligands and receptors. Unique, species-specific features of either the ligands and/or the receptors will help to further define structurally and functionally important domains of the receptor. The resulting findings may allow the design of therapeutic agents for the treatment of hypercalcemia due to hyperparathyroidism or the HHM syndrome, and could have important implications for the understanding and the therapeutic approach towards osteoporosis. In Aim III, the above tools will be used to identify PTH-producing cells in pre- and post-metamorphosis anurans where parathyroid glands first appear, and in fish which have PTH, yet lack anatomically defined parathyroid glands. Furthermore, PTH and PTHrP will be evaluated in vivo in fish, target organs containing receptors for both ligands will be identified by Northern blot analysis, in situ hybridization and immunohistochemistry. The role of PTHrP (and PTH) in early embryogenesis will be evaluated in Xenopus laevis oocytes, blastomeres, and whole embryos by assessing mesoderm induction and patterning.