Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells
Hewang Li, … , Robin A. Felder, Pedro A. Jose
Hewang Li, … , Robin A. Felder, Pedro A. Jose
Published May 8, 2008
Citation Information: J Clin Invest. 2008;118(6):2180-2189. https://doi.org/10.1172/JCI33637.
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Research Article Cardiology

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

Abstract

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Authors

Hewang Li, Ines Armando, Peiying Yu, Crisanto Escano, Susette C. Mueller, Laureano Asico, Annabelle Pascua, Quansheng Lu, Xiaoyan Wang, Van Anthony M. Villar, John E. Jones, Zheng Wang, Ammasi Periasamy, Yuen-Sum Lau, Patricio Soares-da-Silva, Karen Creswell, Gaétan Guillemette, David R. Sibley, Gilbert Eisner, Robin A. Felder, Pedro A. Jose

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Figure 6

The AT1R is ubiquitinated, and the ubiquitination of the AT1R is initiated at the plasma membrane.

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The AT1R is ubiquitinated, and the ubiquitination of the AT1R is initiat...
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.