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 (D 1 R and D 5 R) stimulate adenylyl cyclase (AC) activity, whereas the D 2 -like receptors (D 2 , D 3 and D 4 ) inhibit AC activity. D 1 R, but not the D 5 R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D 1 R and D 5 R 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 D 1 R and D 5 R. 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-hD 5 R (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 (1μM/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-β-cyclodextrin decreased basal AC activity in HEK-D 1 R (− 94.5 ± 2.0% of control) and HEK-D 5 R 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 D 1 R than D 5 R, in agreement with the greater colocalization of AC5/6 with D 1 R than D 5 R 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 D 1 R- and D 5 R-mediated AC signaling. [ABSTRACT FROM AUTHOR]