CLONING OF THE PARATHYROID HORMONE RECEPTOR CDNA

甲状旁腺激素(PTH)在调节骨骼和 矿物质代谢，但对甲状旁腺激素的机制知之甚少 作用于其受体。我的总体目标是确定分子 甲状旁腺素受体作用的基础。这项建议的第一个目的是 扩展我们对受体的生化特征，通过定义 一个主要的蛋白水解性裂解位点的功能意义 产生一种明显解偶联的受体，通过更好地定义 糖基化在受体功能中的作用。我们的主要目标是， 然而，是甲状旁腺素受体基因的分离。这将使我们能够 来推断它的氨基酸序列，这反过来将提供一个合乎逻辑的 旨在阐明甲状旁腺激素结构基础的研究框架 受体功能。将使用两种互补的克隆策略。 首先，我们将使用最近开发的PTH受体单抗 免疫筛选克隆在Lambda中的肾和骨细胞cDNA文库 表达载体。第二种方法是使用聚合酶链 聚合酶链式反应(PCR)和与之对应的简并寡核苷酸 密切相关的G(S)偶联受体的高度保守结构域 从骨骼和肾脏中选择性地扩增出甲状旁腺素受体基因。 具有适当核苷酸和推导出的氨基酸序列的克隆 将被用来重新筛选原始的cDNA文库以获得近 全长克隆，从中可以获得整个PTH受体序列 推断出来的。一旦完成，我们将通过以下方式验证克隆的序列 全长克隆的真核表达及鉴定 特异性125I-BPTH(1-34)结合和甲状旁腺激素反应性腺苷环化酶 活动。这些目标的完成将使今后能够澄清 利用位点定向研究甲状旁腺素受体功能的结构基础 诱变和构建受体嵌合体以确定 负责配体结合和G(S)偶联的特定结构域。 这些研究的成功完成将为我们提供对 甲状旁腺激素受体功能的分子基础 G蛋白偶联受体家族。

Parathyroid hormone (PTH) has a central role in regulating bone and mineral metabolism, yet little is known of the mechanisms underlying PTH action at its receptor. My overall goal is to determine the molecular basis of PTH receptor action. The first aim of this proposal is to extend our biochemical characterization of the receptor, by defining the functional significance of a major proteolytic cleavage site that generates an apparently uncoupled receptor, and by better defining the role of glycosylation in receptor function. Our major objective, however, is the isolation of the PTH receptor cDNA. This will enable us to deduce its amino acid sequence, which in turn will provide a logical framework for studies designed to elucidate the structural basis of PTH receptor function. Two complementary cloning strategies will be used. First, we will use recently developed PTH receptor monoclonal antibodies to immunoscreen renal and bone cell cDNA libraries cloned in a lambda expression vector. A second approach will be to use the polymerase chain reaction (PCR) and degenerate oligonucleotide primers corresponding to highly conserved domains in closely related G(s)-coupled receptors to selectively amplify the PTH receptor cDNA from bone and kidney cDNA. Clones with the appropriate nucleotide and deduced amino acid sequence will be used to rescreen the original cDNA libraries to obtain near full-length clones, from which the entire PTH receptor sequence may be deduced. Once this is done we will validate the cloned sequence by eukaryotic expression of the full-length clones and assessment of specific 125I-bPTH(1-34) binding and PTH responsive adenylate cyclase activity. Completion of these aims will permit the future elucidation of the structural basis for PTH receptor function, using sitedirected mutagenesis and the construction of receptor chimeras to determine the specific domains responsible for ligand binding and G(s)-coupling. Successful completion of these studies will provide new insights into the molecular basis of function not only for the PTH receptor, but also for the family of G-protein coupled receptors.