Figure 7.

CaMKII and Epac1 interact with the carboxyl terminus of β₁-ARs. (A) Tg mice with cardiac-specific overexpression of mouse WT–β₁-AR (WT–β₁-AR Tg) were administered with saline (NS) or 5 mg/kg ISO by i.v. infusion for 5 min. LV homogenates were immunoprecipitated with anti-Flag antibody. Immunoprecipitated proteins were analyzed by Western blotting using the specific antibodies. Stimulation with ISO increased the amount of CaMKII, Epac1, and β-arrestin binding with β₁-AR in hearts. (B and C) HEK-293 cells were transiently transfected with CaMKII-δC and Flag-Epac1, along with either HA–β₁-AR (B) or HA–β₂-AR (C). Serum-starved cells were stimulated with either 10 µM ISO or 5 µM 8-CPT for 10 min at 37°C. β-ARs were immunoprecipitated with anti-HA beads and immunoblotted for associated Epac1, β-arrestin, and CaMKII. ISO stimulation resulted in significant recruitment of Epac1, β-arrestin, and CaMKII to β₁-AR, whereas only β-arrestin recruited to the β₂-AR complex. (D) HEK-293 cells were transiently transfected with either β₁(1–7)/β₂(CT) or β₂(1–7)/β₁(CT) chimeric receptors, along with CaMKII-δC and Flag-Epac1. Serum-starved cells were stimulated with 10 µM ISO for 10 min at 37°C. The receptor chimeras were immunoprecipitated with anti-HA beads and immunoblotted for associated Epac1, β-arrestin, and CaMKII. The C-tail of β₁-AR rather than β₂-AR interacted with CaMKII and Epac1 after ISO stimulation. (E) HEK-293 cells were transfected with either control or β-arrestin1/2 siRNA. HEK-293 cells were then transfected with HA–β₁-AR along with CaMKII-δC and Flag-Epac1. Serum-starved cells were stimulated with 10 µM ISO for 10 min at 37°C. β₁-ARs were immunoprecipitated with anti-HA beads and immunoblotted for associated Epac1, β-arrestin, and CaMKII. Depletion of β-arrestin1/2 inhibited ISO-induced recruitment of CaMKII and Epac1 to the β₁-AR complex. Data illustrated in A–E are representative of five independent experiments. IB, immunoblot; IP, immunoprecipitation.