CLONING OF THE PARATHYROID HORMONE RECEPTOR CDNA

甲状旁腺激素（PTH）在调节骨和 矿物质代谢，但很少有人知道PTH的机制 对其受体的作用。 我的总体目标是确定分子 PTH受体作用的基础。 这项建议的第一个目的是 扩展我们的受体的生化特性，通过定义 一个主要的蛋白水解切割位点的功能意义， 产生一个明显的非偶联受体，并通过更好地定义 糖基化在受体功能中的作用。 我们的主要目标， 然而，是PTH受体cDNA的分离。 这将使我们 推导出它的氨基酸序列，这反过来又会提供一个逻辑的 旨在阐明PTH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受体单克隆抗体 免疫筛选肾和骨细胞cDNA文库克隆在λ 表达载体 第二种方法是使用聚合酶链 反应（PCR）和简并寡核苷酸引物， 与G（s）偶联受体密切相关的高度保守结构域， 从骨和肾cDNA选择性扩增PTH受体cDNA。 具有适当核苷酸和推导的氨基酸序列的克隆 将用于重新筛选原始cDNA文库，以获得近 全长克隆，可以从其获得整个PTH受体序列。 推导 一旦完成，我们将通过以下方式验证克隆序列： 全长克隆的真核表达和 特异性125 I-bPTH（1-34）结合和PTH应答性腺苷酸环化酶 活动 这些目标的完成将允许未来的阐明 PTH受体功能的结构基础，使用定点 突变和受体嵌合体的构建，以确定 负责配体结合和G（s）偶联的特异性结构域。 这些研究的成功完成将提供新的见解， 不仅是PTH受体的分子基础， 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.