Kim Brandon H, Wang Fang I, Pereverzev Alexey, Chidiac Peter, Dixon S Jeffrey
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry; and Bone and Joint Institute; The University of Western Ontario, London, Canada.
ACS Pharmacol Transl Sci. 2019 May 22;2(3):155-167. doi: 10.1021/acsptsci.8b00053. eCollection 2019 Jun 14.
The parathyroid hormone 1 receptor (PTH1R) is a Class B G-protein-coupled receptor that is a target for osteoporosis therapeutics. Activated PTH1R couples through Gs to the stimulation of adenylyl cyclase. As well, β-arrestin is recruited to PTH1R leading to receptor internalization and MAPK/ERK signaling. Previously, we reported that the agonist potency of PTH1R is increased in the presence of extracellular ATP, which acts as a positive allosteric modulator of PTH signaling. Another nucleotide, cytidine 5'-monophosphate (CMP), also enhances PTH1R signaling, suggesting that ATP and CMP share a moiety responsible for positive allostery, possibly ribose-5-phosphate. Therefore, we examined the effect of extracellular sugar phosphates on PTH1R signaling. cAMP levels and β-arrestin recruitment were monitored using luminescence-based assays. Alone, ribose-5-phosphate had no detectable effect on adenylyl cyclase activity in UMR-106 rat osteoblastic cells, which endogenously express PTH1R. However, ribose-5-phosphate markedly enhanced the activation of adenylyl cyclase induced by PTH. Other sugar phosphates, including glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-bisphosphate, also potentiated PTH-induced adenylyl cyclase activation. As well, some sugar phosphates enhanced PTH-induced β-arrestin recruitment to human PTH1R heterologously expressed in HEK293H cells. Interestingly, the effects of glucose-1-phosphate were greater than those of its isomer glucose-6-phosphate. Our results suggest that phosphorylated monosaccharides such as ribose-5-phosphate contain the pharmacophore for positive allosteric modulation of PTH1R. At least in some cases, the extent of modulation depends on the position of the phosphate group. Knowledge of the pharmacophore may permit future development of positive allosteric modulators to increase the therapeutic efficacy of PTH1R agonists.
甲状旁腺激素1受体（PTH1R）是B类G蛋白偶联受体，是骨质疏松症治疗药物的作用靶点。激活的PTH1R通过Gs偶联以刺激腺苷酸环化酶。此外，β-抑制蛋白被招募到PTH1R，导致受体内化和MAPK/ERK信号传导。此前，我们报道在细胞外ATP存在的情况下PTH1R的激动剂效力增加，ATP作为PTH信号传导的正变构调节剂。另一种核苷酸，5'-单磷酸胞苷（CMP），也增强PTH1R信号传导，表明ATP和CMP共享负责正变构的部分，可能是5-磷酸核糖。因此，我们研究了细胞外糖磷酸酯对PTH1R信号传导的影响。使用基于发光的测定法监测cAMP水平和β-抑制蛋白募集。单独的5-磷酸核糖对内源性表达PTH1R的UMR-106大鼠成骨细胞中的腺苷酸环化酶活性没有可检测到的影响。然而，5-磷酸核糖显著增强了PTH诱导的腺苷酸环化酶激活。其他糖磷酸酯，包括1-磷酸葡萄糖、6-磷酸葡萄糖、6-磷酸果糖和1,6-二磷酸果糖，也增强了PTH诱导的腺苷酸环化酶激活。此外，一些糖磷酸酯增强了PTH诱导的β-抑制蛋白募集到在HEK293H细胞中异源表达的人PTH1R。有趣的是，1-磷酸葡萄糖的作用大于其异构体6-磷酸葡萄糖。我们的结果表明，磷酸化单糖如5-磷酸核糖含有PTH1R正变构调节的药效基团。至少在某些情况下，调节程度取决于磷酸基团的位置。药效基团的知识可能允许未来开发正变构调节剂以提高PTH1R激动剂的治疗效果。
