Substance P and PACAP activate dural mast cells through MRGPRX2, not NK1R or PAC 1 /VPAC receptors

Aim Substance P (SP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are mast-cell-activating peptides implicated in headache and of growing therapeutic interest, but the receptors through which they act on mast cells remain poorly defined, since each peptide can signal through several receptors. Here we set out to determine which of these receptors are expressed by dural mast cells. Methods We reanalysed a pan-organ single-cell mast-cell atlas, profiling the candidate receptors for both peptides, corroborated the findings at the protein level in an independent quantitative proteomic atlas of human skin and tested them directly in a single-cell dataset of human dural mast cells. Results In mouse connective tissue mast cells (CTMCs), Mrgprb2 was expressed in 58% of cells versus 0-0.6% for the canonical receptors. In human CTMCs, MRGPRX2 (5.9%) exceeded every canonical receptor (≤ 0.7%). Given the known single-cell dropout of low-abundance receptor transcripts, this enrichment was confirmed at the protein level, where MRGPRX2 was mast-cell-enriched and the canonical receptors undetected. A similar pattern was found in human dural mast cells, where the canonical receptors were essentially absent (PAC 1 , NK2R and NK3R undetected, NK1R 0.7%) and MRGPRX2 was the only receptor detected (2.2%). Conclusion Of the candidate receptors for SP and PACAP, only MRGPRX2 is expressed by CTMCs and dural mast cells at appreciable levels. MRGPRX2 is the likely route through which both peptides act on these cells and represents a potential drug target for primary headaches.