The carboxyl terminus of β₁-AR but not β₂-AR is necessary for CaMKII activation and conformational changes of β-arrestin. (A) Structure of chimeric β₁/β₂-ARs. Dark and light lines indicate the peptide sequences derived from β₁- and β₂-AR, respectively. The β₁(1–7)/β₂(CT) represents β₁-AR containing the carboxyl terminus of β₂-AR, whereas β₂(1–7)/β₁(CT) represents β₂-AR containing the carboxyl terminus of β₁-AR. (B) HEK-293 cells were transiently transfected with WT–β₁-AR, β₁(1–7)/β₂(CT), β₂(1–7)/β₁(CT), or WT–β₂-AR, along with CaMKII-δC and Flag-Epac1. Serum-starved cells were stimulated with 10 µM ISO at 37°C. The CaMKII activation was quantified, expressed as fold increase over nonstimulated WT–β₁-AR, and shown as mean ± SEM (n = 5). Increased ISO-mediated CaMKII activation was observed in cells expressing WT–β₁-AR and β₂(1–7)/β₁(CT) chimeric receptors. *, P < 0.01 versus nonstimulation. (C) HEK-293 cells were transiently transfected with WT–β₁-AR, β₁(1–7)/β₂(CT), β₂(1–7)/β₁(CT), or WT–β₂-AR with β-arrestin2 double brilliance biosensor (Luc–β-arrestin2–YFP). The BRET ratio, which indicated the conformational change of β-arrestin2, was determined. ΔBRET was defined as the ISO stimulated ‒ nonstimulated BRET ratio, and data are shown as mean ± SEM (n = 5). *, P < 0.05 versus WT–β₁-AR. (D) HEK-293 cells were transiently transfected with WT–β₁-AR, WT–β₂-AR, β₂/V2, or V2R, along with CaMKII-δC and Flag-Epac1. Serum-starved cells were stimulated with 10 µM ISO or vasopressin at 37°C. The CaMKII activation was quantified, expressed as fold increase over nonstimulated WT–β₁-AR, and shown as mean ± SEM (n = 5). *, P < 0.01 versus nonstimulation. (E) HEK-293 cells were transiently transfected with CaMKII-δC and Flag-Epac1, along with HA–β₁-AR, HA–β₂-AR, HA-β₂/V2, or HA-V2R. Serum-starved cells were stimulated with either 10 µM ISO or vasopressin at 37°C. β-ARs were immunoprecipitated with anti-HA beads and immunoblotted for associated Epac1, β-arrestin, and CaMKII (n = 5). IP, immunoprecipitation; T-CaMKII, total CaMKII.