PTH-Induced Endocytosis of TbetaRII/PTH1R as a Complex

描述（由申请人提供）：甲状旁腺激素是哺乳动物钙稳态和骨代谢的主要调节剂。其信号系统已成为开发新的骨质疏松症合成代谢治疗方法的主要目标。然而，PTH在骨骼中发挥作用的确切机制尚不完全清楚。在确定甲状旁腺激素下游信号传导机制方面已经取得了重大进展，但详细的下游信号传导机制似乎并不能解释甲状旁腺激素对骨的合成代谢作用。包括PTH1R在内的七种跨膜受体的内吞作用协调不同的信号，并作为细胞的基本组织者，控制许多细胞过程，包括细胞命运的决定。因此，我们开始考虑甲状旁腺激素诱导的内吞作用是否调节和整合其他合成代谢信号。我们的初步数据表明，PTH诱导TGF - II型受体（T - RII）向PTH1R募集，从而促进它们的信号传导，并且这两种受体都被内化为T - RII/PTH1R复合物。免疫沉淀和FRET实验表明形成了一个由T¿RII， PTH1R和PTH组成的三重复合物（1-84）。因此，TGF¿和PTH的信号是协调的。T¿RII的表达增强pth诱导的PTH1R的内吞作用，减少细胞表面PTH1R的数量。因此，cAMP的产生、PKC和ERK1/2活性下调。相反，PTH将T¿RII募集到PTH1R，导致内化不足，抑制了TGF -诱导的Smad信号。因此，我们假设PTH诱导的T¿RII和PTH1R的内吞作用作为一个复合物整合了TGF和PTH转导的信号。因此，PTH对骨的不同作用是由PTH和TGF¿信号的背景以及协同信号的产生来指定的。这样，PTH在引发下游信号的同时，还可以通过诱导T¿RII的内吞作用来调节骨祖细胞的分化、增殖和偶联。该提案分为三个目的：体外信号机制分析，以及在T¿RII和TGF¿1敲除小鼠中分析。在Aim I中，将研究PTH诱导的T¿RII/PTH1R复合物内吞作用对细胞信号传导的影响。PTH1R胞质域被T¿RII磷酸化的位点将被表征。在Aim II中，pth诱导的T¿RII内吞作用对骨重塑的影响将在T¿RII条件敲除小鼠中进行表征。TGF¿1在pth诱导的合成代谢骨形成中耦合骨吸收和形成的作用将在TGF¿1敲除小鼠中进行研究。公共卫生相关性：PTH（1-34）是PTH的n端合成肽，在临床上使用，是唯一可用于治疗骨质疏松症患者的合成代谢剂。甲状旁腺激素配体及其信号传导成分为开发新的骨质疏松症合成代谢治疗方法提供了潜在的主要靶点。然而，目前对下游信号机制的了解状况并不能充分解释甲状旁腺激素对骨的合成代谢作用。在这里，我们提出了解决这个问题的一种范式转变。根据我们的初步数据，我们假设甲状旁腺激素调节其他合成代谢信号，如TGF¿，并协调信号传导过程。我们发现PTH下调TGF -¿信号在调节成骨细胞分化成骨过程中的作用。我们的研究结果的验证可以解释甲状旁腺激素对骨骼的矛盾作用：甲状旁腺激素持续给药的分解代谢作用和间歇给药的合成代谢作用。进一步阐明假设的机制，如本文所提出的，可能导致新的合成代谢疗法的发展。

DESCRIPTION (provided by applicant): PTH is the primary regulator of calcium homeostasis and bone metabolism in mammals. Its signaling system has served as a major target for the development of novel anabolic therapeutic approaches for osteoporosis. However, the exact mechanisms by which PTH exerts its actions in bone are not fully understood. Significant progress has been made in determining the mechanisms of PTH downstream signaling, but the detailed downstream signaling mechanisms do not seem to provide an explanation for the anabolic effects of PTH on bone. Endocytosis of seven-transmembrane receptors, including PTH1R, coordinates different signals and acts as a fundamental organizer of the cell, controlling many cellular processes, including cell fate determination. Thus, we began to consider whether PTH-induced endocycytosis modulates and integrates other anabolic signals. Our preliminary data show that PTH induces recruitment of the TGF¿ type II receptor (T¿RII) to PTH1R, thereby facilitating their signaling, and that both receptors are internalized as a T¿RII/PTH1R complex. Immunoprecipitation and FRET experiments demonstrated formation of a triple complex consisting of T¿RII, PTH1R, and PTH (1-84). As a result, signaling of TGF¿ and PTH is coordinated. Expression of T¿RII enhances PTH-induced endocytosis of PTH1R and reduces the amount of cell surface PTH1R. Consequently, cAMP production, PKC and ERK1/2 activities are down-regulated. Conversely, the recruitment of T¿RII to PTH1R by PTH resulting in under internalization dampened TGF¿-induced Smad signaling. Therefore, we hypothesize that PTH-induced endocytosis of T¿RII and PTH1R as a complex integrates the signals transduced from both TGF¿ and PTH. Thus, the different effects of PTH on bone are specified by the context PTH and TGF¿ signals and the generation of coordinated signals. In this way, while PTH elicits downstream signaling, it also can modulate differentiation, proliferation and coupling of osteoprogenitors through induction of endocytosis of T¿RII. The proposal is organized into three aims: analysis of the signaling mechanisms in vitro, and in T¿RII and TGF¿1 knockout mice. In Aim I, the effects of PTH- induced endocytosis of the T¿RII/PTH1R complex on cellular signaling will be examined. The phosphorylation sites of PTH1R cytoplasmic domain by T¿RII will be characterized. In Aim II, the effects of PTH-induced T¿RII endocytosis on bone remodeling will be characterized in both T¿RII conditional knockout mice. The role of TGF¿1 in coupling bone resorption and formation in PTH-induced anabolic bone formation will be examined in TGF¿1 knockout mice in the Aim III. PUBLIC HEALTH RELEVANCE: PTH(1-34), the N-terminal synthetic peptide of PTH, is used clinically and represents the only anabolic agent that is available for treatment of patients with osteoporosis. PTH ligands and their signaling components provide potential major targets for the development of novel anabolic therapeutic approaches for osteoporosis. However, the current state of knowledge of the downstream signaling mechanisms does not provide an adequate explanation for the anabolic effects of PTH on bone. Here, we propose a paradigm shift in the approach to this problem. Based on our preliminary data, we hypothesize that PTH regulates other anabolic signals, such as TGF¿, and coordinates the signaling processes. We have found that PTH down-regulates TGF¿ signaling in modulating osteoblast differentiation for bone formation. Validation of our findings could account for the paradoxical effects of PTH on bone: a catabolic effect on continuous administration of PTH and an anabolic effect on intermittent administration. Further elucidation of the hypothetical mechanisms, as proposed herein, could result in the development of novel anabolic therapies.