Figure 5.

Effects of Ser/Thr residues on MRGPRX2’s carboxyl-terminus on cell surface expression, SP-induced Ca²⁺ mobilization, and degranulation in transiently transfected RBL-2H3 cells. (A) Snake diagram of secondary structure of MRGPRX2. Each circle represents amino acid residue with one letter code. Solid red backgrounds denote Ser/Thr residues; (B) schematic representation of the carboxyl-terminus of MRGPRX2 (WT) and a phosphorylation-deficient mutant in which all Ser/Thr were replaced with Ala (△ST-MRGPRX2); (C) cell surface expression of WT and △ST-MRGPRX2 was determined by flow cytometry using PE-anti-MRGPRX2 antibody. Representative histograms for WT-MRGPRX2 (black line), △ST-MRGPRX2 (red line), and control untransfected cells (blue line) are shown; (D) cells expressing WT and △ST-MRGPRX2 were loaded with Fura-2 and intracellular Ca²⁺ mobilization in response to SP (1 μM) was determined. Data shown are representative of three independent experiments; (E) cells were exposed to a buffer (control) or SP (1 μM) for 30 min, and β-hexosaminidase release was determined. All data points are the mean ± SEM of at least three experiments performed in triplicate. Statistical significance was determined by a nonparametric t-test. * p ≤ 0.05.