Role of AT1 receptor Ubiquitination in the D5 dopamine receptor‐mediated AT1 receptor degradation

It is known that the angiotensin type I receptor (AT 1 R) is degraded in lysosomes after its binding to angiotensin II. However, in HEK 293 cells overexpressing both human AT 1 R (hAT 1 R) and human D 5 dopamine (hD 5 R) receptors, AT 1 R is degraded in proteasomes after stimulation of the hD 5 R with fenoldopam, a D 5 R agonist (Li et al, FASEB J, 2005, 19: A907). Most proteins destined for proteasome degradation are ubiquitinated, and lysine is the amino acid responsible for ubiquitination. AT 1 R contains many lysine residues, especially in its intracellular domains. The first‐, and third‐ intracellular loops and the C‐terminus of hAT 1 R have ~20% lysine residues. To investigate whether hAT 1 R is ubiquitinated by fenoldopam in hAT 1 R‐hD 5 R HEK 293 cells, cells were stained with anti‐ubiquitin antibody conjugated with Alexa 633; anti‐LAMP I and anti‐p44S 10 conjugated with Alexa 549 were used to detect lysosomes and proteasomes, respectively. Fenoldopam (1μM, 20 min) induced the colocalization of ubiquitin, hAT 1 R, and the proteasome marker p44S 10 , indicating that hAT 1 R may be ubiquitinated by activation of D 5 R, and that the ubiquitinated hAT 1 R is then targeted to proteasomes for subsequent degradation. Ubiquitination of endogenously expressed AT 1 R in rat renal proximal tubule cells following fenoldopam (1μM, 20 min) treatment was determined by immunoprecipitation with anti‐AT 1 R antibody and immunoblotting with anti‐AT 1 R and anti‐ubiquitin antibodies. Taken together, these studies show that ubiquitination is important in the fenoldopam‐stimulated AT 1 R degradation in proteasomes.