STRUCTURAL REQUIREMENTS FOR INTERACTION OF PTH WITH ITS RECEPTOR

甲状旁腺激素在调节血钙中的重要作用 胚胎中甲状旁腺激素相关肽(PTHrP)的水平及其参与 疾病的发展及其作为致病因素的鉴定 与某些恶性肿瘤相关的严重高钙血症，强调 需要彻底了解这些多肽是如何结合和 激活它们的感受器。这些实验的主要目标是 以下是1)识别配基中的关键残基和确定 它们对构象、受体结合和受体的作用 激活，以及2)识别和表征关键残基 配体与之相互作用的受体。预计会有很多人 甲状旁腺素中的残基构成了受体-配体的复杂阵列 互动。我们首先将重点放在PTH中的那些残留 我们的扫描诱变研究表明这一点很重要。我们已经开发出 方法利用克隆的甲状旁腺素受体和Short 合成甲状旁腺素类似物区分关键结合残基 参与了长距离的分子内相互作用或短距离的 相互作用，例如与受体上邻近残基的相互作用。突变 中断远程相互作用将被用来分离基因内， 纠正结合缺陷的第二位点抑制物突变 原发部位突变。这样的突变对将在基因上识别 配基中的两个相互作用的位置。在类似的研究中涉及 甲状旁腺素/甲状旁腺素rP杂交肽，并与M.Weiss博士合作，我们将 将功能的变化与结构的变化相关联 二维核磁共振法。在AIM I中生成的PTH类似物的集合将 用结构上不同的方法筛选受体特异性效应 由定点突变产生的受体或源自 Juppner博士的提案(子项目I)中描述的克隆实验。 我们将绘制受体中决定观察到的 使用嵌合受体和随后的点突变的特异性 分析就像我们对[Arg2]-PTH所做的那样。关键的受体位置将是 通过饱和诱变进行评估，得到的突变体将是 筛选配基特异性效应和激活-构成和 激活缺陷表型。的大的配体结合区 受体，大致被映射到残基1到300之间， 将使用一系列删除来系统地解剖，随后 通过局部随机突变，最后进行点突变分析。至 补充这些遗传方法，我们将获得直接证据 通过物理交联一种特殊的配体-受体相互作用 受体的一系列光衍生物配体。交联体 地点的特征将是用氰化碳和溴化碳碎裂复合体 对SDS-凝胶纯化的标记片段进行测序。这些研究应该 提供大量关于PTH和PTHrP如何 结合并激活它们的共同受体。因此，数据应该为 为合理设计可用于以下目的的新型类似物奠定了基础 从药理上控制这些强效药物的复杂生物效应 多肽。

The vital role of parathyroid hormone (PTH) in regulating serum calcium levels, and the involvement of PTH-related peptide (PTHrP) in embryonic development as well as its identification as the causative agent of severe hypercalcemia associated with certain malignancies, underscore the need for a thorough understanding of how these peptides bind to and activate their receptor. The primary goals of the experiments proposed here are 1) to identify critical residues in the ligand and to determine how they contribute to conformation, receptor binding and receptor activation, and 2) to identify and characterize key residues in the receptor with which the ligand interacts. It is expected that many residues in PTH contribute to the complex array of receptor-ligand interactions. We shall initially focus on those residues in PTH which our scanning mutagenesis studies show to be important. We have developed methods utilizing cells transfected with cloned PTH receptors and short synthetic PTH analogs to distinguish whether critical binding residues are involved in long-range intramolecular interactions or shorter-range interactions, e.g. with nearby residues on the receptor. Mutations which disrupt long-range interactions are to be used to isolate intragenic, second-site suppressor mutations that correct the binding defect of the primary site mutation. Such pairs of mutations will genetically identify two interacting sites in the ligand. In similar studies involving PTH/PTHrP hybrid peptides and a collaboration with Dr. M. Weiss, we shall correlate alterations in function with alterations in structure using the methods of 2D NMR. the collection of PTH analogs generated in aim I will be screened for receptor-specific effects using structurally distinct receptors generated by site-directed mutagenesis or derived from the cloning experiments described in Dr. Juppner's proposal (Subproject I). We shall map the sites in the receptor which determine the observed specificity using chimeric receptors and subsequent point mutation analysis as we have done for [Arg2]-PTH. Key receptor sites will be evaluated by saturation mutagenesis and the resulting mutants will be screened for ligand-specific effects and for activation-constitutive and activation-defective phenotypes. the large ligand-binding region of the receptor, which has been roughly mapped to between residues 1 and 300, will be systematically dissected using a series of deletions, followed by localized random mutagenesis and finally point mutation analysis. to complement these genetic approaches, we shall obtain direct evidence of specific ligand-receptor interactions by physically cross-linking a series of photoderivatized ligands to the receptor. The cross-linked sites will be characterized by fragmenting the complex with CNBr and sequencing the SDS-gel-purified label fragment. These studies should provide a substantial amount of new information about how PTH and PTHrP bind to and activate their common receptor. The data should thus lay the groundwork for the rational design of novel analogs which can be used to pharmacologically control the complex biologic effects of these potent peptides.