GRK2-mediated receptor phosphorylation and Mdm2-mediated β-arrestin2 ubiquitination drive clathrin-mediated endocytosis of G protein-coupled receptors

• Published in: Biochemical and biophysical research communications 2020-12, Vol.533 (3), p.383-390
• Main Authors: Zhang, Xiaohan, Zheng, Mei, Kim, Kyeong-Man
• Format: Article
• Language: English
• Subjects: beta-Arrestin 2 - genetics; beta-Arrestin 2 - metabolism; Caveolin 1 - genetics; Caveolin 1 - metabolism; Clathrin - genetics; Clathrin - metabolism; Endocytosis; Endocytosis - genetics; G-Protein-Coupled Receptor Kinase 2 - genetics; G-Protein-Coupled Receptor Kinase 2 - metabolism; Gene Knockdown Techniques; GPCR; GRK2; HEK293 Cells; Humans; Mutation; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-mdm2 - genetics; Proto-Oncogene Proteins c-mdm2 - metabolism; Receptors, Adrenergic, beta-2 - genetics; Receptors, Adrenergic, beta-2 - metabolism; Receptors, Dopamine D2 - genetics; Receptors, Dopamine D2 - metabolism; Signal Transduction; Ubiquitination; β-Arrestin
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.09.030

Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and β-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and β-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/β-arrestins. For this we utilized the following: (i) mutant dopamine D2 receptor and β2 adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, β-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/β-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/β-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of β-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by β-arrestin ubiquitination is a critical cellular event that links GRK2 and β-arrestins to clathrin-mediated endocytosis. A GPCR can undergo both phosphorylation-dependent and –independent endocytosis. Clathrin-mediated and caveolar endocytosis occur in phosphorylation and activation recognition modes, respectively. Ubiquitination of β-arrestin, which occurs in the nucleus [19], mediates its interaction with clathrin. WT, wildtype; Ago, agonist; β-Arr2, β-arrestin2; CHC, calthrin heavy chain; Cav1, caveolin1; Ub, ubiquitin. [Display omitted] •Receptor phosphorylation is determining factor for clathrin-mediated endocytosis.•Receptor phosphorylation mediates the clathrin vs. GRK2/β-arrestin2 interaction.•Caveolin1 interacts with GRK2/β-arrestin2 without need of receptor phosphorylation.•Ubiquitination is responsible for the interaction between β-arrestin2 and clathrin.