Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada; Bone and Joint Institute, The University of Western Ontario, London, Canada.
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada.
Cell Signal. 2018 Jun;46:103-112. doi: 10.1016/j.cellsig.2018.02.015. Epub 2018 Mar 1.
Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and β-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and β-arrestin signaling as well as downstream activation of CREB. CMP - a nucleotide that lacks a high energy bond and does not activate P2 receptors - mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1-34) required to produce a half-maximal cyclic AMP or β-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1-14) and PTH-related peptide (1-34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism.
甲状旁腺激素 (PTH) 激活成骨细胞和其他靶细胞上的 PTH/PTH 相关肽受体 (PTH1R)。细胞的机械刺激，包括成骨细胞，会导致核苷酸（如 ATP）释放到细胞外液中。除了作为能量源外，ATP 还是 P2 受体的激动剂和许多蛋白质的变构调节剂。我们研究了细胞外 ATP 浓度对 PTH1R 信号转导的影响，这些浓度与激活低亲和力 P2X7 受体的浓度相当。通过生物发光报告实时监测 cAMP 水平，并使用基于互补的发光测定法跟踪 PTH1R 募集的 β-arrestin。ATP 明显增强了 cAMP 和 β-arrestin 信号转导以及 CREB 的下游激活。CMP-一种缺乏高能键且不能激活 P2 受体的核苷酸-模拟了 ATP 的这种作用。此外，这种增强作用不受 P2 受体拮抗剂的抑制，包括 P2X7 的特异性阻断剂。因此，核苷酸诱导的信号通路增强与 P2 受体信号无关。ATP 和 CMP 降低了产生最大 cAMP 或 β-arrestin 反应的一半所需的 PTH(1-34)浓度，而最大受体活性没有明显变化。PTH1R 激动剂 PTH(1-14)和 PTH 相关肽 (1-34)也出现了类似的增加效力。这些观察结果表明，细胞外核苷酸增加了激动剂的亲和力、效力或两者兼而有之，并且与受体或密切相关的蛋白质水平的信号转导调节一致。总之，我们的发现确立了 ATP 通过一种迄今为止尚未被认识的变构机制增强 PTH1R 信号转导。
