Tacrolimus could interact directly with MRGPRX2 to active MCs
To detect whether MRGPRX2 is the target of tacrolimus, molecular docking was implemented. Strikingly, tacrolimus can directly bind to MRGPRX2 by GLU164 residue (Figure 5A). FK-DNS, a fluorescently labeled tacrolimus, was constructed (Figure S1), and only when it binds to MRGPRX2 will it show fluorescence. The detailed information about FK-DNS were shown in Supplementary materials. Similar to tacrolimus, FK-DNS was able to activate LAD2 cells to release β hexosaminidase dose-dependently after short-term treatment and has the similar effect on MRGPRX2 expression after long-term treatment (Figure S2).
To explore whether tacrolimus could interact with MRGPRX2 directly or not, LAD2 cells, MRGPRX2-knockdown LAD2 cells and HMC-1 which expresses no MRGPRX2 were employed. Treated with FK-DNS, NC-LAD2 cells showed significant increased intracellular Ca2+concentration, while calcium influx increased slightly and barely in MRGPRX2-knockdown LAD2 cells and HMC-1 respectively (Figure 5B). Through calcium images, we discovered that FK-DNS was able to bind to NC-LAD2 cells after 1-min exposure, which manifesting as weak fluorescence intensity on the cell membrane, while visible fluorescence was not shown in MRGPRX2-knockdown LAD2 cells and HMC-1 groups. After 10-min tacrolimus treatment in LAD2 cells group, we observed intense fluorescence was inside the cell membrane. In MRGPRX2-knockdown LAD2 cells and HMC-1 groups, only slight fluorescence was found inside the membrane for good lipid solubility of tacrolimus (Figure 5B).
Then, we determined to address the mechanism behind this phenomenon, and it performed that the rapid response to tacrolimus made phosphorylated PLCγ1, PKC and ERK1/2 expression increase in LAD2 cells compared with vehicle treated group. Meanwhile, the quantification of their expression had massive significance (Figure 5C).