Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells
J. Clin. Invest. Hewang Li, et al. 118:2180 doi:10.1172/JCI33637 [Go to this article.]

Figure 5
Fen-stimulated AT₁R degradation occurs in proteasomes but not lysosomes. (A) AT₁R/D₅R HEK293 cells treated with Fen (1 μM for 2 h) and either Chlor (100 μM for 3 h) or CLBL (4 μM for 3 h) were pulsed with [³⁵S] Met/Cys and chased with cold amino acids for the indicated times. Shown is 1 autoradiograph of each treatment. Data (mean ± SEM) followed a first-order exponential curve for both treatments. The AT₁R protein half-life was 43.8 min for Fen plus Chlor and 149.5 min for Fen plus CLBL. n = 3. (B) Sub­cellular distribution of AT₁R in AT₁R/D₅R HEK293 cells treated as in A. Green, AT₁R-EGFP; red, lysosomes or proteasomes (detected by Alexa Fluor 633–conjugated anti–LAMP 1 or anti-p44S¹⁰); yellow, colocalization. Scale bars: 10 μm; 2.5 μm (insets). (C) AT₁R/D₅R HEK293 cells were treated with vehicle, Fen (1 μM for 6 h), Chlor (100 μM for 10 h), CLBL (4 μM for 10 h), Chlor plus Fen, or CLBL plus Fen. Nontransfected D₅R HEK293 cells are shown as a control. Right: MFI of AT₁R-EGFP. n = 3. *P < 0.05, **P < 0.001 versus vehicle, ANOVA, Student-Newman-Keuls test. Data are mean ± SEM. (D) Human RPT cells were treated with vehicle, Ang II, or Fen; cell lysates were incubated with control or proteasome-affinity beads for 4 hours at 4°C. After washing, the matrix was suspended in SDS sample buffer, separated by SDS-PAGE, and transferred onto nitrocellulose. Membranes were immunoblotted for either AT₁R or p44S¹⁰. Experiments were repeated twice with similar results.