REGULATION OF THE PTH/PTHRP RECEPTOR

描述：（直接摘自申请书）PTH的多种内分泌作用和PTHrP的旁分泌作用是由配体的局部浓度和靶细胞的反应性决定的。细胞反应性由细胞表面受体的数量、受体将配体结合转化为G蛋白激活的能力以及对G蛋白的远端生物反应决定。本研究将重点关注调控PTH/PTHrP受体反应性的翻译后事件。我们有强大的工具来探测这些过程中涉及的分子机制。这些包括特异性受体抗体、绿色荧光蛋白标记的受体、突变受体（磷酸化缺陷、组成活性、激活缺陷和gq缺陷）文库、甲状旁腺素类似物（ac选择性）和稳定的骨源性细胞系，这些细胞系在连续或间歇性甲状旁腺素治疗中具有不同的分化模式和骨特异性基因的激活。在Aim I中，磷酸化位点将被绘制，受体磷酸化和G蛋白受体激酶在内化和脱敏中的作用将被确定。在Aim II中，突变受体（将结合与激活分离）将用于确定磷酸化、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受体将用于表征与内化PTH/PTHrP受体囊泡相关的细胞蛋白，并检查受体磷酸化在这一过程中的作用。内化和脱敏的生理相关性将在骨源性细胞系中进行研究，这些细胞系对连续治疗和间歇治疗有不同的生物反应模式。我们在Aim IV中提出干扰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或连续给予甲状旁腺激素的反应。

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.