REGULATION OF THE PTH/PTHRP RECEPTOR

描述：(直接取自应用)甲状旁腺素的不同内分泌作用和旁分泌作用由配体的局部浓度和靶细胞的反应性决定。细胞的反应性取决于细胞表面受体的数量，受体将配体结合转化为G蛋白激活的能力(S)，以及对G蛋白的远端生物反应(S)。这项建议将侧重于了解调节PTH/PTHrP受体反应性的翻译后事件。我们有强大的工具来探索这些过程中涉及的分子机制。这些包括特定的受体抗体、绿色荧光蛋白标记的受体、突变受体文库(磷酸化缺陷、结构活性、激活缺陷和GQ缺陷)、甲状旁腺素类似物(AC选择性)和稳定的骨源性细胞系，这些细胞系具有不同的分化和骨特异基因激活模式，以响应持续和间歇的甲状旁腺素治疗。在目标I中，将绘制磷酸化位点图，并将确定受体磷酸化和G蛋白受体激动子在内化和脱敏中的作用。在AIM II中，分离结合和激活的突变受体将被用来确定磷酸化、内化和/或脱敏是否需要结合和/或激活。在AIM III中，绿色荧光蛋白标记的PTH/PTHrP受体将被用来表征与内化的PTH/PTHrP受体囊泡相关的细胞蛋白，并检测受体磷酸化在这一过程中的作用。内化和脱敏的生理学相关性将在对持续和间歇性甲状旁腺激素治疗有不同生物反应模式的骨源性细胞系中进行检测。我们在AIM IV中建议干预这些细胞的脱敏过程，并确定这种干扰如何影响成骨细胞对间歇和持续给予甲状旁腺素的反应。

Description:(Taken directly from the application) The diverse endocrine actions of PTH and paracrine actions of PTHrP are determined by the local concentrations of ligands and the responsiveness of target cells. Cellular responsiveness is determined by the number of receptors at the cell surface, the ability of receptors to convert ligand binding to G protein(s) activation, and the distal biological responses to G protein(s). This proposal will focus on understanding the post-translational events that regulate the responsiveness of the PTH/PTHrP receptor. We have powerful tools for probing the molecular mechanisms involved in each of these processes. These include specific receptor antibodies, green-fluorescent protein-tagged receptors, and libraries of mutant receptors (phosphorylation-deficient, constitutively-active, activation-deficient and Gq-deficient), PTH analogs (AC-selective) and stable bone-derived cell lines with different patterns of differentiation and activation of bone-specific genes in response to continuous versus intermittent PTH treatment. In Aim I the phosphorylation sites will be mapped and the role of receptor phosphorylation and of G protein receptor kineses in internalization and desensitization will be determined. In Aim II, mutant receptors, which dissociate binding from activation, will be used to determine if binding and/or activation is/are required for phosphorylation, internalization and/or desensitization. In Aim III the green fluorescent protein-tagged PTH/PTHrP receptors will be used to characterize the cellular proteins that associate with the internalized PTH/PTHrP receptor vesicles and to examine the role of receptor phosphorylation in this process. The physiological relevance of internalization and desensitization will be examined in the bone-derived cell lines that have different patterns of biologic responses to continuous versus intermittent PTH treatment. We propose in Aim IV to interfere with the desensitization process in these cells and determine how this interference affects the osteoblast's responses to intermittent versus continuous administration of PTH.