Angiotensin II type receptor (AT1R), a heptahelical protein signals stimuli upon binding of natural peptide ligand angiotensin II (Ang II) and the pathogenic non-HLA IgG antibodies (AT1R -IgG). Interactions of ligands with AT1R lead to active or inactive signaling states responsible for different functional outcomes implicating necessity for new tools allowing for studies of receptor plasticity. We developed a yeast model where the functional expression of a single human AT1R is coupled to yeast's growth response in absence of Histidine. First, the human AT1R cDNA was cloned in a yeast expression plasmid and expressed in the appropriate strain. AT1R activation was induced by addition Ang II or AT1R-IgG isolated from patients with transplant vascular pathology. Both, Ang II and AT1R-IgG triggered a dose-dependent increase in yeasts' growth. AT1R-IgG stimulation induced stronger and more sustainable activation of the receptor than Ang II. AT1R-IgG mediated AT1R-activation could only be slightly attenuated but not abolished by pharmacologic receptor blockers – sartanes which in contrast completely blocked Ang II mediated AT1R-activation. Targeted mutagenesis of all relevant extracellular regions and two major disulphide bridges was performed in order to identify receptor regions governing the activation in response to AT1R-IgG. Mutating of one cystein contained in the disulphide bridge connecting transmembrane region 3 and second extracellular loop (ECL2), as well as both targeted and random mutations of ECL2 and ECL1 impressively abolished yeast growth in response to AT1R-IgG. Exchange of AT1R-ECL2 with ECL2 of Endothelin type A receptor – another functional target for non-HLA antibodies had no influence, stressing specificity for AT1R.

We successfully created a model allowing for structural and functional studies of AT1R plasticity applicable for other antibody GPCR targets. Identification of new extracellular targets responsible for AT1R activation holds great potential for development of new interventions preventing transplant pathologies.

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