Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D-1-like receptors (D1R and D5R) stimulate adenylyl cyclase (AC) activity, whereas the D-2-like receptors (D-2, D-3 and D-4) inhibit AC activity. D1R, but not the D5R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D1R and D5R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D-1 or D-5 receptor (HEK-hD(1)R or HEK-hD(5)R) and human renal proximal tubule (hRPT) cells that endogenously express D1R and D5R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD(1)R (84.5 +/- 2.3% of total), HEK-hD5R (68.9 +/- 3.1% of total), and hRPT cells (66.6 +/- 2.2% of total) (P < 0.05, n = 4/group). In HEK-hD(1)R cells, the D-1-like receptor agonist fenoldopam (1 mu M/15 min) increased AC5/6 protein (+17.2 +/- 3.9% of control) in LRs but decreased it in non-LRs (-47.3 +/- 5.3% of control) (P < 0.05, vs. control, n = 4/group). By contrast, in HEK-hD(5)R cells, fenoldopam increased AC5/6 protein in non-LRs (+67.1 +/- 5.3% of control, P < 0.006, vs. control, n = 4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-beta-cyclodextrin decreased basal AC activity in HEK-D1R (-94.5 +/- 2.0% of control) and HEK-D5R cells (-87.1 +/- 4.6% of control) but increased it in hRPT cells (6.8 +/- 0.5-fold). AC6 activity was stimulated to a greater extent by D1R than D5R, in agreement with the greater colocalization of AC5/6 with D1R than D5R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D1R- and D5R-mediated AC signaling. (C) 2014 Elsevier Inc. All rights reserved.