Fig. 1
Characterization of TRAF6-mediated AKT ubiquitination and phosphorylation under receptor desensitization. A HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were treated with 100 nM Quin or 30 nM DA for 5 min, washed three times with warm serum-free medium, and then treated with increasing concentrations of Quin. The cellular levels of cAMP were indirectly evaluated by using the CRE-luciferase reporter gene assay. Quin treatment group was significantly different from the Veh group at concentrations of quinpirole between 10−10 and 10−8 M (**p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3)). B HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were transfected with GFP-AKT and FLAG-TRAF6, then the cells were treated with 30 nM DA or 100 nM Quin for 5 min according to desensitization protocol. Cell lysates were immunoprecipitated with anti-FLAG beads. Co-IP/lysate and IP were immunoblotted with antibodies against GFP or FLAG, respectively. ****p < 0.0001 compared to the vehicle treated group (n = 3). (C-F) Cell lysates were immunoprecipitated with anti-FLAG beads. Co-IP was immunoblotted with antibodies against HA. IPs were immunoblotted with antibodies against p-AKT(T308), p-AKT(S473) and FLAG, respectively. “+” and “w/+” refer to DA/Quin-treated and washed/retreated cells. C HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were transfected with HA-Ub and FLAG-AKT. The cells were treated with 30 nM DA or 100 nM Quin for 5 min according to desensitization protocol. ****p < 0.0001 compared to the corresponding vehicle treated group (n = 3). D CTRL-shRNA and TRAF6-KD cells were co-transfected with D3R, HA-Ub, and FLAG-AKT. The cells were treated with 100 nM Quin for 5 min according to desensitization protocol. ***p < 0.001, ****p < 0.0001 compared to the corresponding vehicle treated group (n = 3). E HEK293 cells were co-transfected with WT-D3R or C147K-D3R, HA-Ub, and FLAG-AKT. ****p < 0.0001 compared to the corresponding vehicle treated group (n = 3). F HEK293 cells were co-transfected with WT-D2R or K149C-D2R, HA-Ub, and FLAG-AKT. ***p < 0.001, ****p < 0.0001 compared to the corresponding vehicle treated group (n = 3)