EVOLUTION OF THE PTH/PTHRP RECEPTOR AND ITS LIGANDS

甲状旁腺激素(PTH)--哺乳动物最重要的调节因子 钙代谢，由甲状旁腺合成和分泌 腺体；这些腺体在鱼中不存在，在或之前首次出现在无尾两栖动物中 在变态过程中。甲状旁腺激素相关肽(PTHrP)，它具有限制N- 与甲状旁腺素同源的末端氨基酸序列是常见的 治疗恶性体液高钙(HHM)综合征。这很正常 人们对其生理意义知之甚少。甲状旁腺激素相关蛋白的表达 在早期胚胎发育期间，以及在多个胎儿和成人组织中， 然而，暗示自体或旁分泌功能是通过 甲状旁腺素受体，由甲状旁腺素和甲状旁腺素共享。配体和它们的 因此，受体可能对人类具有相当重要的作用 发展。进化研究作为一种方法来评估 激素、生长因子及其效应通路的作用(S) 已被证明对各种内分泌/旁分泌系统有用，因此提供 评估甲状旁腺激素和甲状旁腺激素受体的几个独特机会：1)隔离 新型类似物在配体/受体结构/功能分析中的应用 相互作用与功能重要受体的定义 域，2)新的、技术上更容易的工具来研究以下函数 在哺乳动物系统中不同或可能不太明显，3)强大 用于遗传分析的系统，以及4)用于研究 甲状旁腺素受体(和甲状旁腺素)对人类发育的重要性。为了获得洞察力 了解甲状旁腺素的生理功能，以确定甲状旁腺素是如何进化的 进入哺乳动物矿物质离子新陈代谢的主要调节器，并 评估两个配体如何通过共同的 受体，是这项提议的第一个目标。因此，我们建议 为了研究这两种配体的进化史，他们共同的 受体，或可能与一个或另一个配体结合的受体 优先或排他地通过分离编码这些蛋白质的cDNA 来自鸡肉、非洲爪哇和鱼类，随后从 无脊椎动物物种。甲状旁腺素、甲状旁腺素受体及其受体的功能评价 在AIM II中，来自各种物种的共同受体将扩大我们的 关于配体和配体之间的结构/功能关系的知识 感受器。任何一种配体的独特、物种特有的特征 和/或受体将有助于进一步在结构上和 受体的重要功能结构域。由此得出的发现 可允许设计用于治疗的治疗剂 甲状旁腺功能亢进症或HHM综合征引起的高钙血症，并可能 对理解和治疗有重要意义 治疗骨质疏松症的方法。在AIM III中，将使用上述工具 变态前后无尿症患者甲状旁腺素分泌细胞的鉴定 在甲状旁腺首次出现的地方，在有甲状旁腺的鱼类中，至今 缺乏解剖学上定义的甲状旁腺。此外，PTH和PTHrP 将在鱼体内进行评估，靶器官含有受体 这两种配体将通过原位Northern印迹分析进行鉴定 杂交和免疫组织化学。甲状旁腺素受体(PTHrP)和甲状旁腺素在 非洲爪哇卵母细胞的早期胚胎发育将被评估， 通过评估中胚层的诱导和 图案化。

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.