STRUCTURAL REQUIREMENTS FOR INTERACTION OF PTH WITH ITS RECEPTOR

甲状旁腺素对血钙的调节作用 水平，以及胚胎中PTH相关肽（PTHrP）的参与 发展及其作为病原体的鉴定 严重高钙血症与某些恶性肿瘤，强调 需要彻底了解这些肽如何结合， 激活它们的受体。 提出的实验的主要目标 这里有1）识别配体中的关键残基并确定 它们如何影响构象、受体结合和受体 活化，和2）鉴定和表征在所述细胞中的关键残基。 与配体相互作用的受体。 预计许多 PTH中的残基有助于受体-配体的复杂阵列 交互. 我们将首先关注PTH中的那些残留物， 我们的扫描诱变研究显示是重要的。 我们已经开发 方法利用用克隆的PTH受体和短 合成PTH类似物，以区分关键结合残基 参与长程分子内相互作用或短程 在一些实施方案中，所述化合物可以与例如与受体上的邻近残基的相互作用结合。 的突变 破坏长距离相互作用将用于分离基因内， 第二位点抑制突变，其纠正了 一级位点突变 这种成对的突变将从基因上识别 两个相互作用的位点 在类似的研究中， PTH/PTHrP杂合肽以及与M.韦斯，我们 将功能改变与结构改变相关联， 2D NMR方法。 在目的I中产生的PTH类似物的集合将 使用结构上不同的 通过定点诱变产生的或衍生自 克隆实验在Dr. Bennpner的建议中描述（子项目I）。 我们将绘制受体中决定所观察到的 使用嵌合受体和随后的点突变的特异性 我们对[Arg 2]-PTH进行了分析。 关键受体位点将是 通过饱和诱变进行评价，将得到的突变体 筛选配体特异性效应和活化组成性， 活化缺陷表型。 大的配体结合区， 受体，已大致定位于残基1和300之间， 将使用一系列删除进行系统解剖，然后 通过局部随机诱变和最后的点突变分析。 到 补充这些遗传学方法，我们将获得直接的证据， 特异性配体-受体相互作用通过物理交联 一系列的光衍生配体的受体。 交联 位点将通过用CNBr使复合物片段化来表征， 对SDS-凝胶纯化的标记片段进行测序。 这些研究应 提供了大量关于PTH和PTHrP 结合并激活它们的共同受体。 因此，数据应该奠定 为合理设计新型类似物奠定基础， 控制这些强大的生物效应 缩氨酸

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.