Investigation of the fate of type I angiotensin receptor after biased activation

Biased agonism on the type I angiotensin receptor (AT 1 -R) can achieve different outcomes, via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of the AT 1 -R can lead to different regulation and intracellular processing of the receptor. We analyzed β -arrestin binding, endocytosis and subsequent trafficking steps such as early and late phases of recycling of the AT 1 -R in HEK293 cells expressing wild type or biased mutant receptors in response to different ligands. We used Renilla luciferase tagged receptors and yellow fluorescent protein (YFP) tagged β -arrestin2, Rab5, Rab7 and Rab11 proteins in bioluminescence resonance energy transfer (BRET) measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β -arrestin binding and the internalization kinetics of the angiotensin II (AngII)-stimulated AT 1 -R differed from those stimulated by the biased agonists. Similarly, AngII-stimulated wild type AT 1 -R showed differences compared to a biased mutant AT 1 -R (DRY/AAY AT 1 -R) regarding β -arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane PtdIns(4,5) P 2 depletion. Moreover, the stability of the β -arrestin binding is a major determinant of the later fate of the internalized AT 1 -R receptor. We used BRET-based approach to investigate the fate of the receptor in response to stimuli. We used fluorescently-labeled constructs marking the molecular steps during internalization and luciferase-fused receptors in BRET measurements. Our results demonstrate that biased agonists or stimulation of biased receptor with the native AngII (AT 1 -R-DRY/AAY) yields different fates of the activated receptors in terms of β -arrestin binding, internalization and appearance in various intracellular compartments. Our findings also suggest that the intracellular processing of the AT 1 -R is dependent on the type of activation and the affinity of ligand binding to AT 1 -R. Bias ligand bound receptors are unable to couple to G q proteins and activate PLC β show accelerated internalization due to lack of PtdIns(4,5) P 2 hydrolysis in the plasma membrane. In contrast, the degradation and recycling of the internalized receptor is mainly determined by the strength of β -arrestin binding. the localization and distribution of the receptor were analyzed using a Zeiss LSM 710 confocal laser-scanning microscope. Postacquisition data analysis of the confocal images was performed with either the ZEN (Carl Zeiss) or the MetaMorph (Molecular Devices) software. Quantification of the internalization of AT 1 -R-GFP (location in intracellular vesicles, which clearly are separated from the plasma membrane) as function of time calculated from morphometric analysis by MetaMorph software obtained in seven separate experiments.