Human kidney-2 cells harbor functional dopamine D1 receptors that require Giα for Gq/11α signaling

Abstract

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to G s protein in response to the D1R agonist fenoldopam. Since D1R also couples to G q protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to G q and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to G q. Protein kinase C (PKC) and 86rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and G i protein (pertussis toxin) inhibitors and G q/11α siRNA. Furthermore, fenoldopam and angiotensin II increased 35S-GTPγS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in G q/11α-depleted cells. Also, fenoldopam-mediated inhibition of 86rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and G q/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of G q/11α. The immunoprecipitated levels of G q/11α in the membranes were greater in fenoldopam-treated cells, and G iα coimmunoprecipitated with G q/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling G q-mediated downstream signaling in a G i dependent manner.