Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

Abstract

β-arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptorsand 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. Since 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, G q/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 Ndomain. Using improved FlAsH-based 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.The family of G protein-coupled receptors (GPCRs) consists of ~800 members in humans and about 30% of modern drugs target these molecules (1). GPCRs respond to a wide variety of endogenous ligands, including hormones, neurotransmitters, and lipids. Despite their huge diversity, the signal transduction mechanisms of GPCRs share several common features: agonist binding is followed by the activation of a relatively small number of heterotrimeric G proteins, which initiate complex intracellular signaling cascades. Receptor responsiveness to further stimulation is attenuated by a multistep process, called desensitization (2). In case of homologous desensitization, active GPCRs are phosphorylated by GPCR kinases (GRKs) followed by the recruitment of β-arrestin proteins (β-arrestin1 and JBC C o n f i d e n t i a lHeterologous regulation of inactive receptors via β-arrestin 2 β-arrestin2, also known as arrestin-2 and arrestin-3, respectively). β-arrestins uncouple the receptors from G proteins and initiate receptor internalization (3), thereby serving as the key regulators of GPCRs' function. In contrast, heterologous desensitization is mediated by second-messenger activated kinases, such as protein kinase C (PKC), which can phosphorylate active and inactive receptors. Heterologous desensitization was originally thought to be independent of β-arrestins, however, some data have challenged this concept (4-6). In addition to their rol...

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

“…Similarly, inhibition or lack of G q/11 was shown not to affect ßarrestin-dependent internalization of free fatty acid receptor 4 and thyrotropin-releasing hormone (Yu & Hinkle 1999, Alvarez-Curto et al 2016. The involvement of PKC in ß-arrestin recruitment upon H1R activation remains to be further elucidated but is in agreement with the role of ß-arrestins in desensitization of GPCRs phosphorylated by second messenger-activated kinases (Tóth et al 2018, Luo et al 2017, especially in the view of PKC-dependent phosphorylation of H1R (Fujimoto et al 1999, Horio et al 2004). Conversely, ß-arrestin recruitment to AT1 A R and ß2AR was shown to be predominantly dependent on GRK-mediated phosphorylation, and not on PKC or cAMP-dependent protein kinases (Kim et al 2008, Violin et al 2008a).…”

β‐arrestin 2 negatively regulates NOD2 signalling pathway through association with TRAF6 in microglia after cerebral ischaemia/reperfusion injury

“…4 Moreover, we have been proved NOD2 contributed to cerebral injury by NOD2-mediated inflammatory signalling in mice after ischaemic stroke. 7,8 Growing evidence shows that β-arrestin 2 (ARRB2) play a critical regulatory role in the inflammatory response. β-arrestins were adaptor proteins which terminate G protein activation by desensitizing and internalizing G-protein-coupled receptors (GPCRs).…”

“…6 More recently, β-arrestins have been identified as scaffold proteins binding with various target molecules and thus regulating a wide range of biological processes. 7,8 Growing evidence shows that β-arrestin 2 (ARRB2) play a critical regulatory role in the inflammatory response. 9 Li et al reported ARRB2 acts…”

Arrestins: Introducing Signaling Bias Into Multifunctional Proteins

“…Among other things, arrestins were implicated in recruiting signaling proteins to microtubules, 21 in cytochrome C release during apoptosis, 22 focal adhesion dynamics, 23 activation of small GTPases, 24–27 glucose maintenance, 28 and many other cellular functions, 29,30 including even heterologous desensitization of GPCRs phosphorylated by second messenger-activated kinases. 31,32 …”