Mechanisms of activation of the NLRP3 Inflammasome by extracellular calcium ions

细胞外Ca2+离子通过触发G蛋白偶联受体CaSR和GPRC6A，被主要申请人小组确定为单核细胞中促炎细胞效应机制的有效刺激。对于这两种受体，精确的配体结合位点和进入细胞的信号转导途径尚不清楚。申请人未发表的数据表明，对于exCa2+诱导的刺激，磷酸盐离子的存在是绝对必要的。在Ca2+和磷酸盐浓度升高的促炎条件下，可形成磷酸钙预成核簇和磷酸钙晶体。该项目的目的是研究促炎性Ca2+和磷酸盐感知背后的机制，以及离子簇和磷酸钙晶体在这一过程中的作用。所需的实验将使用固定的CaSR敲除和GPRC6A缺陷小鼠模型以及对磷酸敏感、钠依赖的pi -共转运体PiT-1的基因敲除实验进行。这两种gpcr的外结构域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种受体的结构分析。

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Unpublished data of the applicants show that for the exCa2+-induced stimulation, the presence of phosphate ions is an absolut requirement.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.