Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT₁R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT₁R can also be activated by auto-antibodies (AT₁R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT₁R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT₁R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT₁R signaling. AT₁R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects G_q/11 and G_i activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT₁R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT₁R is impeded by binding of AT₁R-Abs. Secondly, exclusive AT₁R-Abs-induced G_q/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT₁R activation of G_i signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of G_i basal activity potentiates proliferation triggered by AT₁R-Abs. Finally, although AT₁R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT₁R and Abs. This current study thus provides extended insights into the molecular action of AT₁R-Abs and associated mechanisms of interrelated pathogenesis.