Angiogenic Host Defense Peptide AG-30/5C and Bradykinin B2 Receptor Antagonist Icatibant Are G Protein Biased Agonists for MRGPRX2 in Mast Cells

Abstract

AG-30/5C is an angiogenic host defense peptide that activates human mast cells (MC) via an unknown mechanism. Using short hairpin RNA-silenced human MC line LAD2 and stably transfected RBL-2H3 cells, we demonstrate that AG-30/5C induces MC degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2). Most G protein-coupled receptors signal via parallel and independent pathways mediated by G proteins and b-arrestins. AG-30/5C and compound 48/80 induced similar maximal MC degranulation via MRGPRX2, which was abolished by pertussis toxin. However, compound 48/80 induced a robust b-arrestin activation as determined by transcriptional activation following arrestin translocation (Tango), but AG-30/5C did not. Overnight culture of MC with compound 48/80 resulted in reduced cell surface MRGPRX2 expression, and this was associated with a significant decrease in subsequent MC degranulation in response to compound 48/80 or AG-30/5C. However, AG-30/5C pretreatment had no effect on cell surface MRGPRX2 expression or degranulation in response to compound 48/80 or AG-30/5C. Icatibant, a bradykinin B 2 receptor antagonist, promotes MC degranulation via MRGPRX2 and causes pseudoallergic drug reaction. Icatibant caused MC degranulation via a pertussis toxin-sensitive G protein but did not activate b-arrestin. A screen of the National Institutes of Health Clinical Collection library led to the identification of resveratrol as an inhibitor of MRGPRX2. Resveratrol inhibited compound 48/80-induced Tango and MC degranulation in response to compound 48/80, AG-30/5C, and Icatibant. This study demonstrates the novel finding that AG-30/5C and Icatibant serve as G protein-biased agonists for MRGPRX2, but compound 48/80 signals via both G protein and b-arrestin with distinct differences in receptor regulation.

Mas-related G protein–coupled receptor X2 and its activators in dermatologic allergies

“…37 Besides activation of G ai protein-and G aq protein-coupled signaling, recent studies further suggest a MRGPRX2-mediated activation of b-arrestin. 31 Interestingly, the MRGPRX2 ligand C48/80 induced robust b-arrestin activation and receptor internalization, whereas the MRGPRX2 ligands icatibant and the angiogenic host defense protein Ag-30/5C did not, indicating ligand-dependent internalization mechanisms potentially mediated via a differential activation of b-arrestin. 31…”

“…29,30 Unlike other GPCRs (eg, C3aR), MRGPRX2 may be resistant to phosphorylation, internalization, and desensitization following activation by certain, albeit not all, agonists. 15,31 The latter fact may explain the responsiveness of MRGPRX2 to multiple ligands. 3 A single amino acid of the receptor was identified to be critical for binding and activation by its ligands, including pruritogens such as SP.…”

“…21 Codeine induced MRGPRX2 activation in LAD2 cells triggered by phosphorylation of ERK and JNK, but not p38. 37 Inhibitory studies were performed using the ion La 31 , which blocks calcium release-activated calcium channels on the endoplasmic reticulum and plasma membrane. Their results suggest that MRGPRX2 additionally signals via the PTx-insensitive G aq protein.…”

“…136 Several synthetic and humanized AMPs showed antimicrobial activity and induced MRGPRX2-dependent degranulation of LAD2 cells. 31,156 Interestingly, mutated hBD-3 in which all cysteine residues were replaced by serine residues and a shortened LL-37 (comprising residues 17-29) induced MRGPRX2dependent degranulation of LAD2 cells and lacked the lipopolysaccharide-induced blockage of degranulation. 156 Thus, compared with naturally occurring AMPs, these synthetic AMPs potentially exhibit augmented antimicrobial efficiency.…”

“…Whether MRGPRX2 activation triggers receptor internalization is still controversially discussed, since C48/80 provoked b-arrestin-mediated receptor internalization, but LL-37 and AG-30/5C did not. 15,31 In this context, it is likely that the variety of MRGPRX2 agonists have different effects on MRGPRX2 availability, MC activation, released signaling molecules, and immune cell recruitment. Until now, the role of MRGPRX2 agonists in skin diseases has been analyzed only individually; however, it is very likely that allergic conditions are provoked by a ''cocktail'' of MRGPRX2 agonists that may complement or reverse each other's effects.…”