The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor-β-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin-BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that despite β-arrestin2 overexpression, T282M's and C289W's residency with β-arrestin2 in endosomes was greatly reduced, leading to decreased β-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally dependent β-arrestin-mediated MAPK activation as well as endosomal receptor-β-arrestin complex stabilization in the mitogenic response of AT1R.
Keywords: ERK1/2; G protein-coupled receptor (GPCR); MAPK; angiotensin II; angiotensin II type 1 receptor (AT1R); arrestin; bioluminescence resonance energy transfer (BRET); endocytosis; extracellular-signal-regulated kinase (ERK); mitogen-activated protein kinase; naturally occurring variants; trafficking.
© 2020 Cao et al.