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).