Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2

Abstract

Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2.

Keywords: MRGPRX2; amino acid residues; ligand-receptor interactions; mast cells; substance P.

Figure 1

SP stimulates LAD2 degranulation and release of proinflammatory mediators. LAD2 cells were activated with SP for 30 min (1 μM and 5 μM). For comparison, LAD2 were sensitized with IgE (0.5 μg/mL) overnight and challenged with streptavidin (0.1 μg/mL) for 30 min. β-hex release (A) and histamine release (B) were measured as described in Methods. CCL2 (C) and TNF (D) production after SP activation for 24 h was measured by ELISA. Data is presented as mean ± SEM (n = 4, ****p < 0.0001).

Figure 2

Analyis of MRGPRX2 and FcεRIα surface expression by flow cytometry. (A) LAD2 cells were treated with 5 μM SP and MRGPRX2 expression was assessed after 60 min (MRGPRX2 is purple curve; isotype control is red curve). (B) MRGPRX2 expression after 1, 5, 10, 30 and 60 min activation with SP as measured by mean fluorescence intensity (MFI). (C) Expression of FcεRIα after activation with SP as in (B) (FcεRIα is the blue curve; isotype control is the red histogram). (D) FcεRIα expression after 1, 5, 10, 30 and 60 min activation with SP as measured by MFI. Data is presented as mean ± SEM (n = 3, *p < 0.01, **<p 0.05, ***p < 0.001, ****p < 0.0001).

Figure 3

Two configurations of Complex 9 captured during the pulling simulation. Configurations of Complex 9 correspond to the center of mass distance of 3.2 nm (A) and 5.0 nm (B). MRGPRX2 (colored according to its secondary structure) and SP (red colored) are represented by cartoons (special VMD drawing method). Salt-bridge residues are represented by ball and stick models. Dashed lines denote h-bonds between the proteins.

Figure 4

Release of immune mediators by LAD2 cells stimulated with SP analogs. (A) LAD2 cells were activated with SP analogs (SP1, to SP5) for 30 min, and β-hex release was measured. (B) CCL2, and (C) TNF release were analyzed after activation of LAD2 with SP analogs or SP (5 μM) for 24 h Untreated cells were included as negative controls. Data is presented as mean ± SEM (n = 5 for β-hex release, n = 3 for CCL2 release, and n = 4 for TNF release, *p<0.01, ** <p0.05, ***p<0.001, ****p<0.0001).

Figure 5

Flow cytometry analysis of SP analogs on MRGPRX2 expression in LAD2 cells. Histograms showing MRGPRX2 internalization effect after 60 min SP analogs treatment (A, SP1; C, SP2; E, SP4). LAD2 cells were treated overtime (1 to 60 min) with SP analogs (B, SP1; D, SP2; F, SP4), and MRGPRX2 expression was compared to untreated cells. Data is presented as mean ± SEM (n = 3, *<0.05, **<0.01, ***<0.001, ****<0.0001).

Figure 6

Inhibitory effect of SP analogs on the degranulation of LAD2 cells by the 30 min activation by SP. (A-C) Time dependent effect of SP1 preincubation on the β-hex release from the SP activated LAD2 cells. (D-F) Time dependent effect of SP2 preincubation on the β-hex release from the SP activated LAD2 cells. (G-H) Effect of 180 min SP1 and SP2 preincubation on the histamine release from the SP activated LAD2 cells. Untreated and SP treated β-hex and histamine values are included as controls in all tested conditions. Data is presented as mean ± SEM (n = 4, *p<0.01, **p<0.05, ***p<0.001, ****<0.0001).

Figure 7

Inhibitory effect of SP analogs on the release of preformed and de novo synthesized CCL2 from SP activated LAD2 cells. LAD2 cells were preincubated with SP analogs for 3h and then were activated with SP for the mentioned amount of time. Data showing CCL2 release by the SP analog alone refers to the release of CCL2 due to the preincubation of LAD2 cells with SP analogs, and was measured after the 3h SP analog incubation. (A) Release of preformed CCL2 from the SP activated (30 min) LAD2 cells preincubated with SP1. (C) Release of preformed CCL2 from the SP activated LAD2 cells preincubated with SP2. (E) Release of preformed CCL2 from the SP activated LAD2 cells preincubated with SP4. (B) Release of de novo synthesized CCL2 from the SP activated LAD2 cells preincubated with SP1. (D) Release of de novo synthesized CCL2 from the SP activated LAD2 cells preincubated with SP2. (F) Release of de novo synthesized CCL2 from the SP activated LAD2 cells preincubated with SP4. Untreated and SP treated values are included as controls in all tested conditions. Data is represented as mean ± SEM (n = 3, *p<0.01, **p<0.05, ***p<0.001, ****<0.0001).

Figure 8

Inhibitory effect of SP analogs on the release of preformed and de novo synthesized TNF from SP stimulated LAD2 cells. LAD2 cells were preincubated with SP analogs for 3h and then were activated with SP for the mentioned amount of time. Data showing TNF release by the SP analog alone refers to the release of TNF due to the preincubation of LAD2 cells with SP analogs, and was measured after the 3h SP analog incubation. (A) Release of preformed TNF from the SP activated LAD2 cells preincubated with SP1. (C) Release of preformed TNF from the SP activated LAD2 cells preincubated with SP2. (E) Release of preformed TNF from the SP activated LAD2 cells preincubated with SP4. (B) Release of de novo synthesized TNF from the SP activated LAD2 cells preincubated with SP1. (D) Release of de novo synthesized TNF from the SP activated LAD2 cells preincubated with SP2. (F) Release of de novo synthesized TNF from the SP activated LAD2 cells preincubated with SP4. Untreated and SP treated values are included as controls in all tested conditions. Data is represented as mean ± SEM (n = 3, ****<0.0001).