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

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 D receptor and β 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.