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 6
The AT₁R is ubiquitinated, and the ubiquitination of the AT₁R is initiated at the plasma membrane. AT₁R/D₅R HEK293 cells were treated with vehicle or Fen (1 μM for 5 min). (A) Immunostaining for p44S¹⁰ or Ub (clone P4D1). Distance calibration was based on objective and zoom of the images taken: 1 pixel equals 0.133 μm. Green, EGFP-tagged AT₁R; red, p44S¹⁰; blue, Ub; yellow, colocalization of AT₁R and p44S¹⁰ (green and red); cyan, colocalization of AT₁R and Ub (green and blue); magenta, colocalization of p44S¹⁰ and Ub (red and blue); white, colocalization of AT₁R, Ub, and p44S¹⁰ (red, green, and blue). Scale bars shown for vehicle-treated cells also apply to Fen-treated cells. (B) The image of AT₁R-EGFP (as donor fluorophore) was used for drawing ROIs, which were divided into plasma membrane (rectangles) and cytoplasm (ovals) to determine the spatial interaction between the AT₁R and Ub (clone P4D1, Alexa Fluor 555; as acceptor fluorophore). The pure FRET and 2-dimensional distribution images of energy transfer efficiency (E%) were generated as described in Methods. Graphs show mean ± SEM energy transfer efficiency and distance (r) of ROIs in the plasma membrane (Mem) and cytoplasm (Cyt) in the corresponding AT₁R images processed from 6–8 cells. *P < 0.05 versus vehicle, Student’s t test. Distances beyond experimental limitations (>90 υ) are given a 0 value, indicating no occurrence of FRET.