GRK-mediated receptor phosphorylation followed by association with beta-arrestins has been proposed to be the molecular mechanism involved in the desensitization of G protein-coupled receptors (GPCRs). However, this mechanism does not explain the desensitization of some GPCRs, such as dopamine D-3 receptor (D3R), which does not undergo GRK-mediated phosphorylation. Loss-of-function approaches and mutants of dopamine D-2 receptor and D3R, which exhibit different desensitization properties, were used to identify the cellular components and processes responsible for desensitization. D3R mediated the recruitment of Mdm2 to the cytosol, which resulted in the constitutive ubiquitination of beta-arrestin2 in the resting state. Under desensitization conditions, cytosolic Mdm2 returned to the nucleus, resulting in the deubiquitination of cytosolic beta-arrestins. Deubiquitinated beta-arrestins formed a tight complex with Gbg, thereby sequestering it, causing interference in D3R signaling. In conclusion, this study shows that beta-arrestins, depending on their ubiquitination status, control the G protein cycling by regulating their interactions with Gbg. This is a novel mechanism proposed to explain how certain GPCRs can undergo desensitization without receptor phosphorylation. (C) 2020 Elsevier Inc. All rights reserved.