Anqi Wan
Hydar Ali
| Anqi Wan | Hydar Ali |
Anqi Wan
Hydar Ali
Basic & Translational Sciences, University of Pennsylvania, School of Dental Medicine
Introduction
Mast cells are granulated resident immune cells that are found beneath the epithelia of all vascularized tissues. A novel G protein-coupled receptor, MAS-related GPCR X2 (MRGPRX2) is expressed at high level in human skin MCs but is also present in colon MCs but at low levels. MRGPRX2 is activated by an array of cationic peptides including an endogenous peptide, proadrenomedullin N-terminal 9-20 peptide (PAMP-12) produced by keratinocytes and gut mucosa. Activation of MRGPRX2 by PAMP-12 is associated with ulcerative colitis and allergic contact dermatitis. In addition to coupling to G proteins, MRGPRX2 undergoes phosphorylation resulting in β-arrestin-mediated desensitization and activation of downstream signaling. Two missense MRGPRX2 mutations have been identified (A74S and S284P) that likely affect receptor phosphorylation and β-arrestin-mediated responses. The purpose of this study was to investigate the effects of these variants on MRGPRX2 signaling and MC functions in vitro.
Methods
MRGPRX2 missense variants, A74S and S284P, were generated using site-directed mutagenesis The plasmids encoding MRGPRX2 cDNA, and its missense variants were used to transfect HTLA cells (a HEK293 cell line expressing tTA‐ dependent luciferase reporter and β‐arrestin2‐TEV fusion gene) using lipofectamine carrier. Flow cytometry was used to analyze the cell surface expression of MRGPRX2. The arrestin translocation (Tango) assay was performed to measure β-arrestin recruitment by the receptor to determine transcriptional activation of MRGPRX2. The plasmids were also used to transfect rat basophilic leukemia (RBL–2H3) cells by electroporation. Degranulation assay was performed on transfected RBL cells by exposure to compound 48/80 and PAMP-12.
Results
HTLA cells expressing A74S mutant showed a similar level of β-arrestin recruitment while S284P mutant showed a significant decrease in β-arrestin recruitment compared to the WT receptor regardless of the receptor agonists used. This suggest that while S284P variant showed impaired -arrestin pathway, A74S variant remained unaffected. RBL cells expressing the A74S mutant receptor showed a similar degree of degranulation while the S284P mutant receptor showed substantially less degranulation and the co-expressing A74S, S284P mutant receptor showed about 50% decrease in degranulation than the WT receptor regardless of the receptor agonists used. This suggests that while the S284P variant showed a reduced degranulation pathway, the A74S variant is tolerable.
Conclusion
PAMP-12 activate MCs through the β-arrestin pathway and the G protein pathway. The A74S mutation does not affect the receptor responsiveness to MRGPRX2 agonists, while the S284P mutation does.