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 β 1-AR subtype. To identify the potential domain(s) of β 2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular (β1/β 2-Li3 or the carboxyl terminus (β1/β 2-CT) or both domains (β1/β2-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 β 2-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 β 1-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 Gi with pertussis toxin, but not by a panel of β 1-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 Gi 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.
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