Fig. 9
AKT ubiquitination is required for β2AR desensitization. A CTRL-shRNA and TRAF6-KD cells were transfected with β2AR (1.5 ~ 1.7 pmol/mg protein). Then the cells were pretreated with vehicle or 1 μM ISO for 5 min, washed three times, and treated with increasing concentrations of ISO. The cellular levels of cAMP were evaluated by using the CRE-luciferase reporter gene assay. CTRL-shRNA/ISO group was significantly different from other groups at treatment concentrations of ISO between 10−7 and 10−5 M. ***p < 0.001 compared to “CTRL-shRNA/ Veh” group (n = 3). B HEK293 cells were cotransfected with β2AR, WT-AKT or K8/14R-AKT. Then the cells were pre-treated with vehicle or 1 μM ISO 5 min, washed three times, and treated with increasing concentrations of ISO. The cellular levels of cAMP were evaluated by using the CRE-luciferase reporter gene assay. The WT-AKT/ISO group was significantly different from the WT-AKT/Veh group at isoproterenol concentrations between 3 × 10−7 and 10−5 M (*p < 0.05, **p < 0.01 (n = 3)). The K8/14R-AKT/ISO group was significantly different from the K8/14R-AKT/Veh at isoproterenol concentrations between 10−6 and 10−5 M (#p < 0.05 (n = 3)). C CTRL-shRNA and TRAF6-KD cells were cotransfected with β2AR and GFP-Mdm2. The cells were treated with 1 μM ISO for 2 min, washed, and re-challenged. The colocalization between Mdm2 and DAPI is shown as Pearson’s coefficient. ****p < 0.0001 compared to corresponding “CTRL-shRNA/Veh” group, ####p < 0.0001 compared to “CTRL-shRNA/w+” group (n = 11). The scale bar represents 10 μm. D Diagram showing the molecular mechanisms of AKT ubiquitination in homologous desensitization of GPCRs: Under desensitization conditions (repeated agonist exposure), TRAF6 interacts with importin β1 and enters the nucleus in a Src-dependent manner. Within the nucleus, TRAF6-mediated the ubiquitination of AKT, thereby promoting its the translocation and activation. Phosphorylated AKT subsequently phosphorylates Mdm2, which is in complex with the receptor, Gβγ and β-Arr2. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, leading to the de-ubiquitination of β-Arr2. Deubiquitinated β-Arr2 then forms a complex with Gβγ, leading to the desensitization of GPCRs