Abstract
β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-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 β-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 β-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 β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-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 β-arrestins and reveal their novel role in receptor cross-talk.
Highlights
-Arrestins are key regulators and signal transducers of G protein– coupled receptors (GPCRs)
In the case of homologous desensitization, active GPCRs are phosphorylated by GPCR kinases (GRKs) followed by the recruitment of -arrestin proteins (-arrestin1 and -arrestin2, known as arrestin-2 and arrestin-3, respectively). -Arrestins uncouple the receptors from G proteins and initiate receptor internalization [3], thereby serving as the key regulators of GPCRs’ function
It has been demonstrated previously that protein kinase C (PKC) phosphorylation sites in the AT1R C terminus overlap with the GRK-target sites [19, 20]
Summary
It has been demonstrated previously that PKC phosphorylation sites in the AT1R C terminus overlap with the GRK-target sites [19, 20]. We tested whether C-terminal phosphorylation by PKC, in the absence of receptor stimulation, can induce -arrestin binding to the receptor To this end, we performed co-precipitation experiments in HEK 293T cells coexpressing YFP- and biotin acceptor peptide (BAP)-tagged AT1R, -arrestin2–Cerulean, and biotin ligase (BirA). To determine the kinetics of heterologous -arrestin binding in real time, we measured bioluminescence resonance energy transfer (BRET) between Rluc8tagged AT1R and -arrestin2–Venus (Fig. 1b). Both PMA and AngII led to an increase of the BRET signal, reflecting the interaction between AT1R and -arrestin (Fig. 1c). Both the specific PKC inhibitor GF109203X and the broad-spectrum serine–threonine kinase inhibitor staurosporine (used at a concentration that does not inhibit GRKs considerably [23]) prevented the PMA or ␣1AAR stimu-
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