Structural and Functional Mechanisms of PTH-Receptor Signaling

描述(申请人提供)：这项拟议研究的目标是确定甲状旁腺激素(PTH)受体(PTHR)信号机制的结构和细胞基础。PTHR是一种主要的G蛋白偶联受体，调节钙稳态和骨转换，是治疗骨质疏松症最有效的靶点。最近发现的甲状旁腺激素及其类似物在甲状旁腺素内化到早期内体后能够延长G蛋白的活性和cAMP的产生，这极大地改变了我们对甲状旁腺素受体的细胞信号的看法，以及我们研究针对这一受体的药物的方式。GPCR的这种新的和意想不到的行为对于理解最近开发的长效PTH类似物，包括处于临床前开发中的用于治疗甲状旁腺功能减退症的LA-PTH，如何在细胞和小鼠中诱导显著的延长信号(cAMP和钙)反应具有特别重要的意义。PTHR的结构决定因素和负责这些作用的细胞机制尚不清楚，这是识别临床相关类似物的进一步进展的障碍。因此，我们提出了一个研究计划来克服这一障碍。提出了两个具体的目标：目标1通过全长PTHR以及PTHR与PTH或LA-PTH结合的结晶学来确定PTHR作用的结构决定因素。我们首先将重点放在PTH配体不同功能性质的结构基础上，以及可能对内体pH变化敏感的离子相互作用，因为我们的初步发现支持内体pH在持续的PTHR信号转导中的关键作用。这些结构和分子信息将进一步应用于AIM 2，它确定了调节内体PTHR信号的细胞机制，重点是内体中发现的低pH条件在决定PTH-PTHR-G蛋白复合体稳定性中的作用。了解PTH和LA-PTH如何与PTHR结合并在特定的亚细胞位置(内小体)触发信号的结构细节和差异的知识有助于深入了解分子和细胞过程，这可能为治疗提供新的机会。PTHR介导的内体Gs信号的选择性靶向可能比细胞表面信号的全局靶向提供更有效和选择性的治疗。

DESCRIPTION (provided by applicant): The goal of this proposed research is to determine the structural and cellular basis underlying the mechanism of parathyroid hormone (PTH) receptor (PTHR) signaling. The PTHR is a major G protein-coupled receptor (GPCR) that regulates Ca2+ homeostasis and bone turnover and is the most effective therapeutic target for osteoporosis. The recently recognized capacity of PTH and certain PTH analogs to prolong G-protein activity and cAMP production after PTHR internalization into early endosomes has drastically changed how we think about cellular signaling of the PTHR, and how we study drugs that target this receptor. This new and unexpected behavior of a GPCR is of particular relevance for understanding how the recently developed long-acting PTH analogs, including LA-PTH that is in preclinical development for the treatment of hypoparathyroidism, induce remarkable prolonged signaling (cAMP and calcium) responses in cells and in mice. The structural determinants of the PTHR and cellular mechanisms responsible for these actions are not known and this is an obstacle to further progress for identifying clinically relevant analogs. We therefore propose a research program to overcome this obstacle. Two specific aims are proposed: Aim 1 determines the structural determinants of PTHR action through crystallography of full-length PTHR, and PTHR bound to PTH, or to LA-PTH. We will focus initially on the structural basis for the different functional properties of PTH ligands and on ionic interactions likely to be sensitive to pH changes encountered in endosomes because our preliminary findings support a critical role of endosomal pH on sustained PTHR signaling. This structural and molecular information will be further applied to Aim 2, which determines the cellular mechanism regulating endosomal PTHR signaling, focusing on the role of low pH conditions found in the endosome in determining the stability of PTH-PTHR-G protein complexes. Knowledge of structural details and differences of how PTH and LA-PTH bind to the PTHR and trigger signaling in specific subcellular locations (endosomes) provides insights into molecular and cellular processes, which can provide new opportunities for therapy. Selective targeting of PTHR-mediated endosomal Gs signaling might offer more effective and selective treatments than global targeting of cell-surface signaling.