GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision

beta-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that beta-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the beta-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that beta-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for beta-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of beta-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream beta-arrestin-mediated events are directed.

Automated summary

Beta-arrestins are strongly autoinhibited in their basal state but can be activated by phosphorylated receptors and receptor agonism, releasing the beta-arrestin tail and allowing it to assume different conformations. The rate and efficiency of beta-arrestin activation depend on the phosphorylation pattern of the receptor and agonist-promoted receptor activation.