Regulation of D5 dopamine receptor signaling in lipid rafts in HEK‐293 Cells

We tested the hypothesis that the D 5 R is regulated by caveolae in lipid rafts (LRs) using human D5R or D 1 R heterologously expressed in HEK‐293 cells (n=3). The D 5 R protein was localized in LRs; the molecular sizes of D 5 R protein ranged from 45 to 250 kDa. The D 5 Rs co‐fractionated with caveolin‐2β (cav‐2β), flotillin‐ 1 (flo‐1) and flotillin‐2 (flo‐2), G Sα , and several signaling molecules. Disruption of LRs with βCD (2%/1hr) increased basal cAMP accumulation 3‐fold but agonist stimulation with fenoldopam (fen, 5 μM/15 min) decreased cAMP accumulation by 30% (vehicle=10.1 and fen =7.4 pmol/mg protein/min). D 5 R co‐localized and co‐immunoprecipitated with cav‐2β. Fen also increased the amount of cav‐2β associated with D 5 R (vehicle=14.6±8.3, fen=53±4.7, density units [DU]), similar to D 1 R (not shown). However, D 1 R and D 5 R differently regulate LR proteins. Fen increased the amount of flo‐1 associated with cav‐2β (fen=36.6±3.6 vs. vehicle=24.7±2.8 DU) (ANOVA, P<0.05) in HEK‐D 1 R cells while fen increased the amount of flo‐2 associated with cav‐2β (fen=39.7±8.1 vs. vehicle=23.9±3.5 DU) (ANOVA, P<0.05) in HEK‐D 5 R cells. In addition, fen increased tyrosine phosphorylation in HEK‐D 5 R cells. Taken together, D 5 R is mainly distributed in LRs and associated with and regulated by cav‐2β but are differentially associated with flotillin; D 1 R with flo‐1 and D 5 R with flo‐2. D 5 R signaling also involves tyrosine phosphorylation.