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Dopamine D1‐like receptors differently regulate small G proteins in LRs in human embryonic kidney cells heterologously expressing human D1 or D5 receptors (1097.7)
Author(s) -
Yu Peiying,
Yang Yu,
Jose Pedro
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1097.7
Subject(s) - fenoldopam , receptor , hek 293 cells , agonist , immunoprecipitation , g protein , dopamine receptor , biology , microbiology and biotechnology , medicine , endocrinology , chemistry , cell culture , biochemistry , genetics
Small G proteins,e.g.,Rabs (Rab4, Rab5, and Rab11), are involved in G protein‐coupled receptor trafficking, including recycling. We tested the hypothesis that D1‐like receptors (D1R and D5R) differentially regulate the small G proteins Rab4, Rab5, and Rab11 in lipid rafts (LRs) in HEK‐293 cells heterologously expressing either the human D1R or D5R (hD1R or hD5R cells, respectively). In the basal state the Rabs, which co‐fractionated with flotillin‐1, an LR marker protein, were distributed in LR fractions (fractions 2‐6) and nonLR fractions (fractions 7‐12). The D1‐like receptor agonist fenoldopam (1‐5μM/15min), increased Rab4 in LRs in hD1R cells (+38.9±14.8%, vs. control 0.0±4.8 %) and in hD5R cells (+26.9±8.7% vs. control +0.1±3.8 %) (P<0.05, n=4, ANOVA). The effect of fenoldopam on Rab5 was opposite that of Rab4: fenoldopam decreased Rab5 protein in LRs in both cell lines (‐27.4±4.9% hD1R) and (‐19.7±3.0% hD5R) (P<0.05, n=5, ANOVA). Fenoldopam also increased Rab11 protein in LRs in hD1R (+41.6±16.3%) compared to its control (+0.0±0.5%) (P<0.01, n=4, ANOVA) but not in hD5R cells. Moreover, fenoldopam increased Rab11 protein co‐immunoprecipitation with D1R in a time dependent manner which peaked at 1h (22.9±2.5 DU), relative to vehicle‐treatment (9.8±1.9 DU) (P<0.01, n=4, ANOVA). Rab11 protein also co‐immunoprecipitated with D5R but was not affected by fenoldopam. Confocal images corroborated the cell fractionation and co‐immunoprecipitation data: fenoldopam decreased Rab5 in LRs in hD1R and hD5R cells, and increased Rab4 in LRs in hD1R and hD5R cells. Fenoldopam also increased Rab11 protein co‐localization with LR marker protein in LRs in hD1R cells. However, in D5R cells, the D5R and Rab11 co‐localization in LRs was not affected by fenoldopam. We conclude that dopamine D1R and D5R co‐fractionates with Rab4, Rab5, and Rab11 in LRs. These small G proteins may play an important role in D1R and D5R trafficking. Specifically, Rab11 protein may play an important role in D1R recycling. Grant Funding Source : HL023081, HL074940, DK039308