Abstract

It is well established that the β₂-adrenergic receptor (β₂-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related β₁-AR subtype. To identify the potential domain(s) of β₂-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (β₁/β_2-Li3) or the carboxyl terminus (β₁/β_2-CT) or both domains (β₁/β_2-Li3CT) of β₁-AR are replaced by the corresponding parts of the β₂-AR. Using adenoviral gene transfer, we individually expressed these β₁/β₂-AR chimeras in mouse cardiomyocytes lacking both native β₁-AR and β₂-AR (β₁/β₂ double knockout), and examined their possible spontaneous activities. Overexpression of these β₁/β₂-AR chimeras markedly elevated basal cAMP accumulation and myocyte contractility in the absence of agonist stimulation compared with those infected by a control adenovirus expressing β-galactosidase or an adenovirus expressing wild type β₁-AR. These effects were fully reversed by a β₂-AR inverse agonist, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 × 10^-7 M), regardless of inhibition of G_i with pertussis toxin, but not by a panel of β₁-AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate (CGP20712A), betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of β₁-AR is not responsible for the lack of β₁-AR spontaneous activation, although it has been known that the β₁-AR PDZ motif prevents the receptor from undergoing agonist-induced trafficking and G_i coupling in cardiomyocytes. Taken together, the present results indicate that both the third intracellular loop and the C terminus are involved in β₂-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.