PARATHYROID HORMONE AND CALCITONIN

众所周知，靶细胞暴露于甲状旁腺激素 (PTH)在体内或体外降低其随后的腺苷酸环化酶（AC） 对PTH的反应（脱敏）。 在培养的鸡胚骨细胞中 （CCEBC），之前暴露于PTH导致几乎完全脱敏 通过放射性配体结合评估，仅部分缺失PTH受体 测定。 这项提案的长期目标是定义分子 脱敏骨中PTH受体“解偶联”的基础 细胞 具体来说，这些研究旨在验证假设 脱敏CCEBC中的PTH受体在功能上和结构上 改变，使得它们不能将刺激信号传输到AC。 初始实验将确定条件（时间、PTH浓度） 所需的最佳解偶联的PTH受体。 初始目标 是为了达到最大程度的脱敏可能与 PTH受体结合的最小扰动。 这些能力 非偶联受体，相对于正常偶联受体， 与腺苷酸的鸟苷酸依赖性调节蛋白（N） 然后将以两种方式评估环化酶。 首先，将进行比较 外源性鸟苷酸对控制受体和非偶联受体的调节 核苷酸 这将涉及计算机辅助动力学分析 在存在和不存在的情况下， 鸟苷酸。 第二，普通话的结构交互作用 受体和N在膜将通过共价标记的评估 每种组分，然后溶解和色谱分离， 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测定来评估。 受体，然后进行凝胶电泳。 包括在后一种情况中， 研究是评估PTH的磷酸化状态是否 受体在脱敏过程中发生改变。 对这一概念的进一步支持 PTH受体结构或功能的变化与 将通过检查以下因素之间的相关性来获得脱敏 这种作用的逆转和CCEBC的再敏感化。 是 预计这些研究最终将提供一个框架， 了解病理检查中观察到的对PTH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).