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）。机械刺激包括成骨细胞在内的细胞会导致核苷酸（如ATP）释放到细胞外液中。除了作为能源的作用外，ATP还充当P2受体的激动剂和许多蛋白质的变构调节剂。我们研究了与激活低亲和力P2X7受体相当的细胞外ATP浓度对PTH1R信号的影响。使用生物发光报告器实时监测循环AMP的水平，并使用基于互补的发光测定法跟踪将β-arrestin募集到PTH1R中。ATP显着增强了环AMP和β-arrestin信号传导以及CREB的下游激活。CMP-一种缺乏高能键且不会激活P2受体的核苷酸-模仿了ATP的这种作用。此外，增强作用不受P2受体拮抗剂（包括P2X7的特异性阻滞剂）的抑制。因此，核苷酸诱导的信号通路增强与P2受体信号传导无关。ATP和CMP降低了产生最大半数环状AMP或β-arrestin反应所需的PTH浓度（1-34），但最大受体活性无明显变化。与PTH1R激动剂PTH（1-14）和PTH相关肽（1-34）相似，效力也明显提高。这些观察结果表明，细胞外核苷酸增加激动剂亲和力，功效或两者，并且与受体或紧密相关的蛋白质水平上的信号传导调节一致。在一起