FIGURE 1.

The Arg-389 variant of the β₁-adrenoceptor is hyperfunctional with regard to agonist-dependent cAMP formation and hyperphosphorylated. A, cAMP formation in HEK293 cells stably expressing the ADRB1 variants at comparable levels (and non-expressing control (Ctr) cells). Stimulation was carried out with 1 μm NE in 384 wells containing 10,000 cells for the indicated time (n = 4, with 3–4 replicates each). *, p < 0.05; Student's t test. B and C, basal and NE-induced phosphorylation of the ADRB1 variants at position 389. HEK293 cells stably transfected with Arg-389-ADRB1 or Gly-389-ADRB1 were labeled with [³²P]orthophosphate. After stimulation with the agonist, the ADRB1 protein was immunoprecipitated, and incorporated radioactivity was quantified by PhosphorImager analysis. B, representative PhosphorImager blot (lanes 5–10, additional transfection of an siRNA mixture targeting GRK-2, 3, 5, and 6 or a respective scrambled control siRNA). C, quantification of phosphorylation of Arg-389-ADRB1 and Gly-389-ADRB1 with and without NE stimulation for 5 min (n = 5). *, p < 0.05; two-way analysis of variance followed by Bonferroni post test. D, representative Western blots of lysates from cells transfected with siRNA targeted against GRK subtypes. E, quantification of phosphorylation of Arg-389-ADRB1 and Gly389-ADRB1 with and without NE stimulation for 5 min upon knockdown of GRK-2, 3, 5, and 6 (n = 3).