Cationic modifications in gonadotropin-releasing hormone analogues drive mas-related G protein-coupled receptor X2-dependent histamine release through charge interactions

Gonadotropin-releasing hormone (GnRH) analogues are widely used polypeptide therapeutics whose clinical utility is limited by pseudo-allergic reactions mediated through histamine release. This study systematically investigated the correlation between GnRH analogue-induced histamine release and the mast cell receptor Mas-related G protein-coupled receptor X2 (MRGPRX2)-a key mediator of drug-induced pseudo-allergic reactions-sing integrated, in vivo, and structural analyses. In vitro experiments demonstrated that GnRH analogues trigger MRGPRX2-dependent Ca²⁺ mobilization and mast cell degranulation, resulting in dose-dependent increases in β-hexosaminidase, histamine, and tumor necrosis factor α (TNF-α) release. Among the analogues tested, nafarelin exhibited the highest potency, whereas buserelin exhibited the lowest. In vivo, these analogues induced mast cell degranulation and capillary dilation in mouse paw skin, leading to localized pseudo-allergic symptoms (edema and extravasation) that were confirmed to be MRGPRX2-mediated. Molecular docking revealed that the cationic amino acid at position eight of the GnRH analogues bound complementarily to the negatively charged center within the MRGPRX2 ligand-binding pocket, suggesting a mechanistic basis for receptor activation. Functional validation via Arg8-to-Glu substitution in triptorelin significantly attenuated MRGPRX2 activation. Collectively, this work elucidates the MRGPRX2-dependent molecular mechanism underlying GnRH analogue-induced histamine release and provides a foundation for designing safer analogues with reduced adverse effects.