*Purpose: Endothelin-1 type A receptor (ET_AR) exhibits multiligand binding abilities and may signal upon natural ligand Endothelin-1 (ET-1) and non-HLA ET_AR-agonistic IgG (ET_AR-IgG)-mediated stimulation similar to AT₁R-IgG. Development of high-resolution methods allows for structural-functional relationship studies of specific receptor modules involved in the receptor activation. Elucidation of ET_AR activation mechanisms could have broad clinical relevance for vascular transplant pathologies across whole spectrum of solid organ Transplantation.

*Methods: To distinguish the activation mediated by the natural ligand or the agonistic antibodies, we developed a GPCR activation assay relying on solely expressed human ET_AR in yeasts and signalling reporter assays for down-stream effectors in mammalian cells. ET_AR activation was induced by either by natural ligand, ET-1 or by ET_AR-IgG isolated from patients with vascular pathology.

*Results: Both, ET-1 and ET_AR-IgG triggered a dose-dependent activation. ET_AR-IgG induced a stronger activation of the receptor than ET-1, implicating their high biologic potency. ET_AR antagonist blocked completely ET_AR activation induced by ET-1, but only partially ET_AR activation mediated by the IgG isolated from patients. Intact first (ECL1) or second extracellular loop (ECL2) were instrumental for G_12/13 signalling. ECL3 mutation led to a constitutive activation of the receptor. Deletion of the N-terminal part of the receptor showed that this domain is involved in the receptor activation mediated by ET_AR-IgG. We successfully created models allowing for structural and functional studies of molecular architecture modules appreciating ET_AR receptor plasticity, which helped us to define the role of the specific extracellular domains.

*Conclusions: ET_AR exhibit unique mode of GPCR targeting that differs from the similar AT₁R-IgG. Better understanding of the molecular mechanisms responsible for ET_AR activation holds great potential for refining mechanisms of rejection and more precise diagnostic and therapeutic developments.

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