钙敏感受体(CaSR),在甲状旁腺和肾脏中大量表达,在钙稳态中起着核心作用。此外,CaSR发挥多模态作用,包括炎症,肌肉收缩,和骨骼重塑,在其他器官和组织中。CaSR的多种功能由许多内源性和外源性配体介导,包括钙,氨基酸,谷胱甘肽,Cinacalcet,和etelcalcetide,在CaSR中具有不同的结合位点。然而,评估配体与CaSR相互作用的策略仍然有限。这里,我们开发了一种基于谷胱甘肽的光亲和探针,DAZ-G,分析配体与CaSR的结合。我们表明DAZ-G与CaSR中的氨基酸结合位点结合,并充当CaSR的正变构调节剂。氧化和还原的谷胱甘肽和苯丙氨酸有效地与DAZ-G缀合到CaSR竞争,而钙,Cinacalcet,和etelcalcetide具有协同作用。一个意想不到的发现是,咖啡因有效地与DAZ-G\与CaSR的共轭竞争,并充当CaSR的正变构调节剂。用于CaSR活化的咖啡因的有效浓度(<10μM)在血浆中通过普通咖啡因消耗容易获得。我们的报告证明了一种新的CaSR化学探针的实用性,并发现了咖啡因的新蛋白质靶标,这表明摄入咖啡因可以调节CaSR的多种功能。
The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione,
cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium,
cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G\'s conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 μM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.