Mechanisms of activation of the NLRP3 Inflammasome by extracellular calcium ions

细胞外钙离子被牵头申请者小组确定为单核细胞促炎细胞效应机制的有力刺激，其作用是通过触发G蛋白偶联受体CaSR和GPRC6A来发挥作用。对于这两种受体，精确的配体结合部位和进入细胞的信号转导途径尚不清楚。申请者的未发表数据表明，对于exCa~(2+)诱导的刺激，磷酸根离子的存在是绝对必需的。然而，在钙和磷浓度升高的促炎条件下，可以形成磷酸钙预成核团和磷酸钙晶体。该项目的目的是研究促炎性钙和磷酸盐感知背后的机制，以及离子簇和钙磷酸盐晶体在这一过程中的作用。所需的实验将使用CaSR基因敲除和GPRC6A缺乏的小鼠模型，以及磷酸盐敏感、钠依赖的PI-Co转运体PIT-1的基因敲除实验。这两个GPCRs的胞外区和全长蛋白将在体外表达，用于配体结合和受体激活的功能研究，以及两个受体的结构分析。

Extracellular Ca2+ ions were identified by the group of the lead applicant as potent stimuli of pro-inflammatory cellular effector mechanisms in monocytes, which act through triggering of the G protein coupled receptors CaSR and GPRC6A. For both receptors, the precise ligand binding sites and signal transduction pathways into the cell are not known.Unpublished data of the applicants show that for the exCa2+-induced stimulation, the presence of phosphate ions is an absolut requirement. Under such pro-inflammatory condition of increased Ca2+ and phosphate concentrations, however, calcium phosphate pre-nucleation cluster and calcium phosphate crystals can develop. The aim of the project is to investigate the mechanisms behind the pro-inflammatory Ca2+ and phosphate sensing, as well as the role of ion clusters and calcium phosphate crystals in this process.The required experiments will be performed using a floxed CaSR knockout and a GPRC6A deficient mouse model as well as genetic knockdown experiments of the phosphate sensitive, sodium dependent Pi-Co-transporter PiT-1. The ectodomains and full length protein of the two GPCRs will be expressed in vitro for functional studies of ligand binding and receptor activation as well for structural analysis of both receptors.