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

ABSTRACT: ?-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ?-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ?-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 ?-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 ?-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 ?-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ?-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ?-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-?-arrestin interaction, but also governs the structural rearrangements within ?-arrestins. Furthermore, we found that ?-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 ?-arrestins and reveal their novel role in receptor cross-talk.

SUBMITTER: Toth AD

PROVIDER: S-EPMC5777260 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Tóth András D AD Prokop Susanne S Gyombolai Pál P Várnai Péter P Balla András A Gurevich Vsevolod V VV Hunyady László L Turu Gábor G

The Journal of biological chemistry 20171116 3

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 ...[more]

PMID: 29146594

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Project description:The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.

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

Publications

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

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