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Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors
Fang-Tsyr Lin1, Wei Chen, Sudha Shenoy
1Howard Hughes Medical Institute and Departments of Medicine and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710, USA.
Abstract:
Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.
Insights
Beta-arrestin2 phosphorylation regulates its interaction with clathrin, impacting G protein-coupled receptor internalization. This distinct mechanism, mediated by casein kinase II, parallels beta-arrestin1 regulation.
Area of Science:
- Cellular Biology
- Molecular Pharmacology
- Signal Transduction
Background:
- Beta-arrestins are key regulators of G protein-coupled receptor (GPCR) signaling, mediating desensitization and internalization.
- Beta-arrestin1 phosphorylation by ERKs at Ser-412 affects its clathrin association and receptor internalization function.
- Understanding beta-arrestin2's regulatory mechanisms is crucial for elucidating GPCR trafficking pathways.
Purpose of the Study:
- To investigate the phosphorylation sites and regulatory mechanisms of beta-arrestin2.
- To determine the role of beta-arrestin2 phosphorylation in its interaction with clathrin and GPCR internalization.
- To compare the phosphorylation pathways of beta-arrestin1 and beta-arrestin2.
Main Methods:
- Site-directed mutagenesis of beta-arrestin2 phosphorylation sites (Thr-383, Ser-361).
- Assessment of beta-arrestin2 association with clathrin and the beta(2)-adrenergic receptor.
- Analysis of beta(2)-adrenergic receptor internalization following isoproterenol stimulation.
- Identification of the kinase responsible for beta-arrestin2 phosphorylation at Thr-383.
Main Results:
- Beta-arrestin2 is predominantly phosphorylated at Thr-383 and Ser-361.
- Isoproterenol stimulation leads to beta-arrestin2 dephosphorylation.
- Mutating phosphorylation sites to aspartic acid reduces beta-arrestin2-clathrin association and beta(2)-adrenergic receptor internalization.
- Casein kinase II, not ERKs, mediates beta-arrestin2 phosphorylation at Thr-383.
- Beta-arrestin2 binding and desensitization of the beta(2)-adrenergic receptor remain unaltered by phosphorylation site mutation.
Conclusions:
- Phosphorylation/dephosphorylation of beta-arrestin2 regulates its clathrin-mediated internalization of the beta(2)-adrenergic receptor, similar to beta-arrestin1.
- Distinct kinases (casein kinase II for beta-arrestin2, ERKs for beta-arrestin1) control arrestin phosphorylation.
- These findings highlight conserved yet distinct phosphorylation-dependent mechanisms governing arrestin function across the family.