PARATHYROID HORMONE AND CALCITONIN

培养的鸡胚胎成骨样细胞(OB)暴露于剂量 甲状旁腺激素(PTH)导致基本完全 甲状旁腺素刺激的环磷酸腺苷产生减少的脱敏作用 比细胞表面甲状旁腺素结合率低于50%。因此，损失了 细胞表面甲状旁腺素受体不能完全解释 观察到的脱敏现象表明，细胞表面的一小部分 脱敏OB的甲状旁腺素受体与环磷酸腺苷解偶联 制作。使用最近开发的单抗(MAb) 我们实验室免疫沉淀甲状旁腺素受体，我们已经获得了 初步证据表明OB中的PTH受体是磷酸化的，并且 OB暴露于甲状旁腺素后迅速(1分钟)增加受体 在细胞最多的时候(30分钟)达到最大的磷酸化 不敏感的。我们的长期目标是检验这一假设 OB血浆中功能性甲状旁腺素受体的稳态水平 膜受它们的磷酸化水平调节， 甲状旁腺素效应的调节剂可能使用磷酸化- 去磷酸化机制以可变地抑制或增强甲状旁腺素的作用， 分别进行了分析。本提案的具体目标旨在 提供多行证据来支持或拒绝这一点 假设。 除了少数例外，我们有相当多的经验 执行本文件中建议的工作所需的主要技术 申请。我们选择了同源现象 以脱敏为框架研究甲状旁腺激素的意义 OB受体的条件下的受体磷酸化 甲状旁腺激素激动剂或甲状旁腺素拮抗剂未被占用或被占用。此外， 甲状旁腺素受体单抗将用于免疫沉淀磷酸化甲状旁腺素 感受器。这些程序应该会提高我们的口译能力 结果通过提供生物学和免疫学的证据 专一性。 我们和其他实验室的调查应该会导致 在可预见的未来克隆甲状旁腺素受体。因此，它将是 可能对特定突变的影响进行研究 甲状旁腺素受体功能上的磷酸化位点。成功完成 将构成本申请中提出的研究的基础 用于这些分子研究的设计，并应有助于提供 为解释他们的结果提供了重要的生物学视角。

Exposure of cultured chick embryonic osteoblast-like cells (OB) to doses of parathyroid hormone (PTH) that cause essentially complete desensitization of PTH-stimulated cyclic AMP production decreases specific cell surface PTH binding by less than 50%. Thus, the loss of cell surface PTH receptors does not entirely account for the magnitude of desensitization observed, suggesting that a fraction of cell surface PTH receptors in desensitized OB are uncoupled from cyclic AMP production. Using monoclonal antibodies (MAbs) developed recently in our laboratory to immunoprecipitate the PTH receptor, we have obtained preliminary evidence that the PTH receptor in OB is phosphorylated and that exposure of OB to PTH rapidly (1 min) increases receptor phosphorylation to a maximum at a time (30 min) when cells are maximally desensitized. Our long-term objective is to test the hypothesis that the steady-state level of functional PTH receptors in the OB plasma membrane is regulated by their level of phosphorylation and that modulators of PTH effects might employ a phosphorylation- dephosphorylation mechanism to variably dampen or enhance PTH action, respectively. The Specific Aims of the present proposal are designed to provide multiple lines of evidence that either support or reject this hypothesis. With few exceptions, we have had considerable experience with the principal technology needed to perform the work proposed in this application. We have selected the phenomenon of homologous desensitization as a framework to investigate the significance of PTH receptor phosphorylation under conditions in which OB receptors are unoccupied or occupied with PTH agonist or PTH antagonist. Furthermore, PTH receptor MAbs will be used to immunoprecipitate phosphorylated PTH receptors. These procedures should enhance our ability to interpret results by providing evidence of both biologic and immunologic specificity. Investigations in our and other laboratories should result in the cloning of the PTH receptor in the foreseeable future. It will thus be possible to perform studies of the influence of specific mutations of phosphorylation sites on PTH receptor function. Successful completion of the studies proposed in the present application will form the basis for the design of these molecular investigations and should help provide important biologic perspective for the interpretation of their results.