EVOLUTION OF THE PTH/PTHRP RECEPTOR AND ITS LIGANDS

PTH/PTHRP 受体及其配体的进化

• 批准号: 6104978
• 负责人: HARALD JUPPNER
• 金额: $ 26.72万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6104978
• 关键词: Xenopus; Xenopus oocyte; alternatives to animals in research; biochemical evolution; embryogenesis; hormone receptor; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; metamorphosis; molecular cloning; northern blottings; parathyroid gland; parathyroid hormone related protein; parathyroid hormones; peptides; protein structure function; receptor binding; tissue /cell culture; trout /salmon

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.

甲状旁腺激素（PTH），哺乳动物的最重要调节剂 钙代谢，由甲状旁腺合成和分泌 腺体；这些在鱼中不存在，首先出现在Anurans中或 在变态过程中。 与PTH相关的肽（PTHRP），其有限 与PTH的末端氨基酸序列同源性通常是负责的 对于恶性肿瘤（HHM）综合征的体液高钙血症。 它正常 生理意义知之甚少。 PTHRP的表达 在早期胚胎发生以及在多个胎儿和成人组织中， 但是，建议通过 PTHRP和PTH共享的受体。 配体及其 因此，受体可能对人类非常重要 发展。 进化研究是一种评估的方法 激素，生长因子及其效应途径的功能具有 被证明对各种内分泌/旁分泌系统有用，因此提供 评估PTH和PTHRP的几个独特机会：1）隔离 用于配体/受体的结构/功能分析的新型类似物的 相互作用和功能上重要的受体的定义 域，2）新颖，技术上更容易研究的工具 在哺乳动物系统中不同或可能不太明显，3）强大 遗传分析的系统，以及4）模型系统来研究 PTHRP（和PTH）对于人类发展的重要性。 获得见解 进入PTHRP的生理功能，以确定PTH如何发展 进入哺乳动物矿物离子代谢的主要调节剂，并 评估两种配体如何通过共同的行动多样化 受体，是该提议的第一个目标。 因此，我们提出了建议 目的是研究两个配体的进化史，它们共同 受体或可能结合一种或另一种配体的受体 优先或仅通过隔离编码这些蛋白质的cDNA 来自鸡肉，Xenopus laevis和鱼，随后从 无脊椎动物。 PTH，PTHRP及其的功能评估 在AIM II中，来自各种物种的常见受体将扩大我们的 了解配体之间的结构/功能关系 受体。 两种配体的独特，特定于物种的特征 和/或受体将有助于进一步定义结构和 受体的功能重要域。 最终的发现 可以允许设计治疗剂治疗 由于甲状旁腺功能亢进或HHM综合征引起的高钙血症，可以 对理解和治疗具有重要意义 骨质疏松症的方法。 在AIM III中，将使用上述工具 鉴定产前和后变形的产生PTH的细胞Anurans 甲状旁腺首次出现的甲状旁腺，以及有pth的鱼 缺乏解剖学上定义的甲状旁腺。 此外，PTH和PTHRP 将在FIVO中评估鱼类，目标器官，其中包含受体 两种配体将通过北印迹分析确定，原位 杂交和免疫组织化学。 PTHRP（和PTH）在 早期胚胎发生将在爪蟾卵母细胞中进行评估， 通过评估中胚层诱导和 图案。