Investigation of the Fate of Type I Angiotensin Receptor after Biased Activation

Abstract

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 AT 1 -R can lead to different regulation and intracellular processing of the receptor. We analyzed b-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT 1 -R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged b-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer 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. b-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT 1 -R differed from those stimulated by the biased agonists. Similarly, angiotensin IIstimulated wild-type AT 1 -R showed differences compared with a biased mutant AT 1 -R (DRY/AAY AT 1 -R) with regards to b-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 phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the b-arrestin binding is a major determinant of the later fate of the internalized AT 1 -R receptor.

Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology

“…Biased AT 1 R agonists accelerated receptor internalization compared with ANG II stimulation. This is due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion (1019).…”

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

“…It has been shown previously that receptor-β-arrestin interaction may regulate the receptor's intracellular processing (47), so we hypothesized that PKC-induced β-arrestin drives intracellular receptor trafficking differently compared to the fully engaged form. To test this idea we used bystander BRET measurements between AT1R-Rluc8 and one of the intracellular vesicle markers, Venus-Rab4, Venus-Rab5, Venus-Rab7 or VenusRab11, as described previously (48,49). Rab5 is located in the early endosomes, Rab4 and Rab11 in the fast and slow recycling vesicles, respectively, whereas Rab7 is found in late endosomes, which fuse with lysosomes (46).…”

“…Cerulean-tagged versions of these mutants and wild type V2R were generated by replacing Sluc to Cerulean using AgeI/NotI restriction enzymes. Venus-Rab4, Venus-Rab7 and Venus-Rab11 were created by replacing EYFP to monomeric Venus in YFP-Rab4, YFP-Rab7 and YFP-Rab11 constructs (48). To generate Rluc8-β-arrestin2, the coding sequence of rat β-arrestin2 was subcloned from YFP-β-arrestin2 (72) after Rluc8 with a short linker sequence (RSRAQACTR) into pRluc8-C1.…”

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins

“…This method was successfully used to follow the intracellular trafficking route of GPCRs from the PM to different intracellular compartments (18,19). To investigate the dynamic changes of the membrane localization of peripheral membrane proteins that bind the PM, and follow their intracellular fates, we utilized the same approach with some modifications.…”