Mdm2-Mediated Ubiquitination Plays a Pivotal Role in Differentiating the Endocytic Roles of GRK2 and Arrestin3

Upon activation of certain G protein-coupled receptors, Mdm2 promotes the ubiquitination of both GRK2 and arrestin3. Similar to arrestin3, GRK2 ubiquitination was associated with its endocytic activity and proteasomal degradation. Ubiquitination of GRK2 was essential for arrestin3 ubiquitination, and vice versa. Cellular components involved in arrestin3 ubiquitination, including Gβγ, clathrin, and 14-3-3η, were also necessary for GRK2 ubiquitination. Additionally, the arrestin-biased signaling pathway contributed to the ubiquitination of both GRK2 and arrestin3. By employing Mdm2-knockdown cells alongside GRK2 and arrestin3 mutants deficient in ubiquitination sites, as well as receptors lacking phosphorylation sites, we established that the ubiquitinated forms of GRK2 and arrestin3 facilitate clathrin-dependent endocytosis, whereas non-ubiquitinated GRK2 and arrestin3 are responsible for caveolar and a distinct third endocytic pathway, respectively. In the context of clathrin-mediated endocytosis, arrestin3's interaction with clathrin and GRK2's interaction with the β2-adaptin subunit of adaptor protein complex 2 were critical. These findings suggest that GRK2 and arrestin3 ubiquitination are mutually dependent, with their ubiquitination states determining their roles in distinct endocytic pathways.