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

Abstract

We tested the hypothesis that the D5R is regulated by caveolae in lipid rafts (LRs) using human D5R or D1R heterologously expressed in HEK‐293 cells (n=3). The D5R protein was localized in LRs; the molecular sizes of D5R protein ranged from 45 to 250 kDa. The D5Rs co‐fractionated with caveolin‐2β (cav‐2β), flotillin‐ 1 (flo‐1) and flotillin‐2 (flo‐2), GSα, 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). D5R co‐localized and co‐immunoprecipitated with cav‐2β. Fen also increased the amount of cav‐2β associated with D5R (vehicle=14.6±8.3, fen=53±4.7, density units [DU]), similar to D1R (not shown). However, D1R and D5R 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‐D1R 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‐D5R cells. In addition, fen increased tyrosine phosphorylation in HEK‐D5R cells. Taken together, D5R is mainly distributed in LRs and associated with and regulated by cav‐2β but are differentially associated with flotillin; D1R with flo‐1 and D5R with flo‐2. D5R signaling also involves tyrosine phosphorylation.