Abstract

β-arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT₁R), to promote receptor desensitization and internalization. The AT₁ R is a class B GPCR that recruits both β-arrestin1 and β-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. β-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) α-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of β-arrestin1 and β-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type β-arrestins. After angiotensin II stimulation, both wild-type and β-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all β-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type β-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and β-arrestin mutants retained a capacity to interact with the AT₁R, although the interaction with the mutants was less stable. Finally, wild-type and mutant β-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in β-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.