Wang Shujie, Kundu Dooti, Zhang Xiaohan, Tian Xinru, Peng Lulu, Kim Kyeong-Man
Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 61186, Republic of Korea.
School of Pharmaceutical Sciences, Guizhou University, Guiyang 550025, China.
Int J Mol Sci. 2025 Mar 31;26(7):3238. doi: 10.3390/ijms26073238.
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.
在某些G蛋白偶联受体激活后，Mdm2促进GRK2和抑制蛋白3的泛素化。与抑制蛋白3相似，GRK2的泛素化与其内吞活性和蛋白酶体降解有关。GRK2的泛素化对抑制蛋白3的泛素化至关重要，反之亦然。参与抑制蛋白3泛素化的细胞成分，包括Gβγ、网格蛋白和14-3-3η，对GRK2的泛素化也必不可少。此外，偏向抑制蛋白的信号通路有助于GRK2和抑制蛋白3的泛素化。通过使用Mdm2敲低细胞以及缺乏泛素化位点的GRK2和抑制蛋白3突变体，以及缺乏磷酸化位点的受体，我们确定GRK2和抑制蛋白3的泛素化形式促进网格蛋白依赖性内吞作用，而非泛素化的GRK2和抑制蛋白3分别负责小窝和另一种不同的第三条内吞途径。在网格蛋白介导的内吞作用中，抑制蛋白3与网格蛋白的相互作用以及GRK2与衔接蛋白复合物2的β2-衔接蛋白亚基的相互作用至关重要。这些发现表明GRK2和抑制蛋白3的泛素化相互依赖，它们的泛素化状态决定了它们在不同内吞途径中的作用。
