PARATHYROID HORMONE AND CALCITONIN

众所周知，靶细胞暴露于甲状旁腺激素 体内或体外甲状旁腺激素(PTH)降低其随后的腺苷环化酶(AC) 对甲状旁腺素的反应(脱敏)。在培养的鸡胚骨细胞中 (CCEBC)，事先接触PTH可导致几乎完全脱敏 根据放射性配基结合的评估，PTH受体仅部分丢失 化验。这项提议的长期目标是定义分子 脱敏骨骼中甲状旁腺素受体“解偶联”的基础 细胞。具体地说，这些研究旨在检验这一假设 脱敏CCEBC中的PTH受体在功能和结构上 被改变，以至于它们不能向交流传输刺激信号。 初步实验将确定条件(时间、PTH浓度) 甲状旁腺素受体最佳解偶联所需。最初的目标 是实现最大程度的脱敏 甲状旁腺素受体结合的最小扰动。这些人的能力 非偶联受体，相对于正常偶联受体，相互作用 与腺苷酸鸟苷依赖的调节蛋白(N)结合 然后，将通过两种方式对Cyclase进行评估。首先，我们将进行比较 外源性鸟苷对控制受体和解偶联受体的调节作用 核苷酸。这将涉及到计算机辅助的动力学分析 有无CCEBC膜上甲状旁腺激素受体结合的研究 鸟嘌呤核苷酸。第二，PTH的结构相互作用 膜上的受体和N将通过共价标记进行评估 每个组分，然后溶解和层析在 非变性系统。此外，PTH的结构变化 脱敏后的受体将通过光亲和性联用来评估 受体，然后进行凝胶电泳。包括在后一种情况下 研究是对甲状旁腺素的磷酸化状态是否 受体在脱敏过程中会发生变化。进一步支持这一概念 甲状旁腺素受体结构或功能的改变是有因果关系的 将通过检查以下各项之间的相关性来获得脱敏 这种作用的逆转和CCEBC的再增敏。它是 预计这些研究最终将为 从病理学角度理解甲状旁腺激素引起的骨骼反应改变 体内状态(例如，维生素D缺乏、皮质类固醇过量)。

It is well established that exposure of target cells to parathyroid hormone (PTH) in vivo or in vitro decreases their subsequent adenylate cyclase (AC) response to PTH (desensitization). In cultured chick embryonic bone cells (CCEBC), prior exposure to PTH results in nearly complete desensitization with only partial loss of PTH receptors, as assessed by radioligand binding assays. The long-range goal of this proposal is to define the molecular basis for this "uncoupling" of the PTH receptor in desensitized bone cells. Specifically, these studies are designed to test the hypothesis that PTH receptors in desensitized CCEBC are functionally and structurally altered so that they are unable to transmit stimulatory signals to AC. Initial experiments will determine the conditions (time, PTH concentration) required for optimal uncoupling of the PTH receptor. The initial objective is to achieve the greatest degree of desensitization possible with the minimum perturbation of PTH-receptor binding. The ability of these uncoupled receptors, relative to normally coupled receptors, to interact with the guanyl nucleotide-dependent regulatory protein (N) of adenylate cyclase will then be assessed in two ways. First, comparisons will be made of the regulation of control and uncoupled receptors by exogenous guanyl nucleotide. This will involve computer-assisted analysis of the kinetics of PTH-receptor binding in membranes from CCEBC in the presence and absence of guanyl nucleotides. Secondly, the structural interaction of PTH receptors and N in the membrane will be assessed via covalent labeling of each component, followed by solubilization and chromatography in non-denaturing systems. In addition, structural changes in the PTH receptor upon desensitization will be assessed by photoaffinity laleling of the receptor, followed by gel electrophoresis. Included in these latter studies is an evaluation of whether the phosphorylation state of the PTH receptor is altered during desensitization. Further support for the notion that changes in the structure or function of PTH receptors relate causally to desensitization will be obtained by examining the correlation between the reversal of such effects and resensitization of CCEBC. It is anticipated that these studies will ultimately provide a framework for understanding the altered skeletal response to PTH seen in pathologic states in vivo (e.g., vitamin D deficiency, corticosteroid excess).