G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation
The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tai...
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MDPI AG
2023-10-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/13/10/1552 |
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| author | Marta Sánchez-Soto Noelia M. Boldizsar Kayla A. Schardien Nora S. Madaras Blair K. A. Willette Laura R. Inbody Christopher Dasaro Amy E. Moritz Julia Drube Raphael S. Haider R. Benjamin Free Carsten Hoffman David R. Sibley |
| author_facet | Marta Sánchez-Soto Noelia M. Boldizsar Kayla A. Schardien Nora S. Madaras Blair K. A. Willette Laura R. Inbody Christopher Dasaro Amy E. Moritz Julia Drube Raphael S. Haider R. Benjamin Free Carsten Hoffman David R. Sibley |
| author_sort | Marta Sánchez-Soto |
| collection | DOAJ |
| description | The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation. |
| first_indexed | 2024-03-10T21:23:44Z |
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| id | doaj.art-a4efa938f10740ff9782ac55fdd0a957 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T21:23:44Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-a4efa938f10740ff9782ac55fdd0a9572023-11-19T15:50:52ZengMDPI AGBiomolecules2218-273X2023-10-011310155210.3390/biom13101552G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor PhosphorylationMarta Sánchez-Soto0Noelia M. Boldizsar1Kayla A. Schardien2Nora S. Madaras3Blair K. A. Willette4Laura R. Inbody5Christopher Dasaro6Amy E. Moritz7Julia Drube8Raphael S. Haider9R. Benjamin Free10Carsten Hoffman11David R. Sibley12Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAInstitut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, GermanyInstitut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, GermanyMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAInstitut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, GermanyMolecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USAThe D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation.https://www.mdpi.com/2218-273X/13/10/1552GRKD<sub>2</sub> receptorphosphorylationβ-arrestin |
| spellingShingle | Marta Sánchez-Soto Noelia M. Boldizsar Kayla A. Schardien Nora S. Madaras Blair K. A. Willette Laura R. Inbody Christopher Dasaro Amy E. Moritz Julia Drube Raphael S. Haider R. Benjamin Free Carsten Hoffman David R. Sibley G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation Biomolecules GRK D<sub>2</sub> receptor phosphorylation β-arrestin |
| title | G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation |
| title_full | G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation |
| title_fullStr | G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation |
| title_full_unstemmed | G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation |
| title_short | G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation |
| title_sort | g protein coupled receptor kinase 2 selectively enhances β arrestin recruitment to the d sub 2 sub dopamine receptor through mechanisms that are independent of receptor phosphorylation |
| topic | GRK D<sub>2</sub> receptor phosphorylation β-arrestin |
| url | https://www.mdpi.com/2218-273X/13/10/1552 |
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