The angiotensin II (Ang II) type 1 receptor (AT(1)R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT(1)R can also be activated by auto-antibodies (AT(1)R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT(1)R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT(1)R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT(1)R signaling. AT(1)R-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 AT(1)R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT(1)R is impeded by binding of AT(1)R-Abs. Secondly, exclusive AT(1)R-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 AT(1)R 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 AT(1)R-Abs. Finally, although AT(1)R 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 AT(1)R and Abs. This current study thus provides extended insights into the molecular action of AT(1)R-Abs and associated mechanisms of interrelated pathogenesis.
Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation