Parathyroid Hormone Receptor Type 1: Dynamics, Traffic and Recycling

内化G蛋白偶联受体（GPCR）的运输和命运在调节中起着核心作用 他们的功能。虽然GPCR内化的一般途径在大多数成员中是共享的， 然而，这些通路的精确调节机制仍然难以捉摸。一个极端 其实例是I型甲状旁腺激素受体（PTH 1 R）。PTH 1 R展现出非凡的Celland 不同配体对其活性和运输的调节存在组织特异性差异。先前 我们实验室和其他人的工作表明，PTH 1 R是通过与抑制无关的 独立的进程。然而，这些过程的相对贡献和 调节它们的机制在很大程度上仍然是未知的。本应用程序将测试假设， PTH 1 R内化受PTH 1 R与PDZ的相互作用的严格调节（PSD 95，大圆盘， Zo-1）蛋白。这里提出的大量初步工作表明，PTH 1 R的流量是由 PDZ蛋白NHERF 1和Dv 12。该提案将解决这些监管机制 蛋白质在骨细胞模型中发挥其功能。这些研究将使用最先进的 成像（时间推移共聚焦和全内反射显微镜，单分子跟踪，和图像 相关光谱）结合经典的膜生物化学和分子生物学 策略（点突变体的行为分析，配体结合研究，免疫沉淀 技术）。所提出的方法的一个主要创新是，它在很大程度上依赖于对 单独的内吞步骤，以补充整体生化分析。开发的工具和 从这项工作中获得的结论将适用于气相化学还原系列的其他成员。

The traffic and fate of internalized G protein coupled receptors (GPCR) play a central role in the regulation of their function. Although the general pathways of GPCR internalization are shared among most members of the family, the precise mechanisms by which these pathways are regulated remain elusive. An extreme example of this is the Type I parathyroid hormone receptor (PTH1R). The PTH1R exhibits remarkable celland tissue-specific differences regarding the regulation of its activity and traffic by different ligands. Previous work from our laboratory and others has shown that the PTH1R is internalized by a combination of arrestindependent and independent processes. However, the relative contributions of these processes and the mechanisms that regulate them are still largely unknown. This application will test the hypothesis that PTH1R internalization is tightly regulated by the interactions of the PTH1R with PDZ (PSD95, discs-large, Zo-1) proteins. Abundant preliminary work presented here shows that the traffic the PTH1R is regulated by the PDZ proteins NHERF1 and Dvl2. This proposal will address the mechanisms by which these regulatory proteins perform their function in bone cell models. These studies will be conducted using state-of-the-art imaging (time-lapsed confocal and total internal reflection microscopy, single molecule tracking, and image correlation spectroscopy) in combination with classical membrane biochemistry and molecular biology strategies (analysis of the behavior of point mutants, ligand binding studies, immunoprecipitation techniques). A major innovation of the approach proposed is that it relies substantially on the analysis of individual endocytic steps to complement the global biochemical analyses. The tools developed and the conclusions obtained from this work will have applications to other members of the GPCR family.