PTH-Induced Endocytosis of TbetaRII/PTH1R as a Complex

描述(申请人提供)：甲状旁腺素是哺乳动物体内钙稳态和骨代谢的主要调节剂。它的信号系统已经成为开发新的骨质疏松症合成代谢治疗方法的主要靶点。然而，甲状旁腺激素在骨骼中发挥作用的确切机制还不完全清楚。甲状旁腺激素下游信号转导机制的研究已取得显著进展，但详细的下游信号转导机制似乎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，控制着许多细胞过程，包括决定细胞命运。因此，我们开始考虑甲状旁腺素诱导的内吞作用是否调节和整合其他合成代谢信号。我们的初步数据显示，甲状旁腺激素可诱导转化生长因子II型受体(T？RII)募集到PTH1R，从而促进其信号转导，并且这两种受体都内化为T？RII/PTH1R复合体。免疫沉淀和FRET实验证实形成了由T？RII、PTH1R和PTH(1-84)组成的三重复合体。因此，转化生长因子β和甲状旁腺激素的信号转导是协调的。T？RII的表达增强了PTH诱导的PTH1R的内吞作用，减少了细胞表面PTH1R的数量。因此，cAMP生成、PKC和ERK1/2活性下调。相反，PTH向PTH1R募集T？RII导致内化不足，抑制了转化生长因子？诱导的Smad信号转导。因此，我们假设PTH诱导的T？RII和PTH1R的内吞作用是一个复合体，整合了转化生长因子β和甲状旁腺素转导的信号。因此，甲状旁腺激素对骨骼的不同作用是由甲状旁腺激素和转化生长因子信号以及协调信号的产生来确定的。通过这种方式，甲状旁腺激素在诱导下游信号传导的同时，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。该建议分为三个目标：分析体外信号机制，以及T？RII和转化生长因子？1基因敲除小鼠的信号机制。在目标I中，将检测PTH诱导的T？RII/PTH1R复合体的内吞作用对细胞信号的影响。将表征PTH1R胞浆结构域被T？RII的磷酸化位点。在AIM II中，将在两个T？RII条件性基因敲除小鼠中表征PTH诱导的T？RII内吞作用对骨重建的影响。目的III将在转化生长因子1基因敲除小鼠身上研究转化生长因子1在甲状旁腺激素诱导的合成骨形成中耦合骨吸收和形成中的作用。公共卫生相关性：甲状旁腺激素的N端合成肽甲状旁腺素(1-34)被用于临床，是唯一可用于治疗骨质疏松症患者的合成代谢剂。甲状旁腺素配体及其信号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，我们提出了一个解决这个问题的方法的范式转变。根据我们的初步数据，我们假设PTH调节其他合成代谢信号，如转化生长因子β，并协调信号过程。我们发现，在调节成骨细胞分化以促进骨形成的过程中，PTH下调了转化生长因子信号。对我们的发现的验证可以解释甲状旁腺素对骨骼的矛盾效应：连续给药时的分解代谢效应和间歇给药时的合成代谢效应。进一步阐明这里提出的假设机制，可能会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.