Heterologous phosphorylation-induced formation of a stability lock permits regulation of inactive receptors by ß-arrestins.

ABSTRACT

ß-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ß-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ß-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of ß-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes ß-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and ß-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ß-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ß-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-ß-arrestin interaction, but also governs the structural rearrangements within ß-arrestins. Furthermore, we found that ß-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of ß-arrestins and reveal their novel role in receptor cross-talk.