The third intracellular loop and the carboxyl terminus of beta2-adrenergic receptor confer spontaneous activity of the receptor.

It is well established that the beta2-adrenergic receptor (beta2-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related beta1-AR subtype. To identify the potential domain(s) of beta2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (beta1/beta2-Li3) or the carboxyl terminus (beta1/beta2-CT) or both domains (beta1/beta2-Li3CT) of beta1-AR are replaced by the corresponding parts of the beta2-AR. Using adenoviral gene transfer, we individually expressed these beta1/beta2-AR chimeras in mouse cardiomyocytes lacking both native beta1-AR and beta2-AR (beta1/beta2 double knockout), and examined their possible spontaneous activities. Overexpression of these beta1/beta2-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 beta-galactosidase or an adenovirus expressing wild type beta1-AR. These effects were fully reversed by a beta2-AR inverse agonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 x 10-7 M), regardless of inhibition of Gi with pertussis toxin, but not by a panel of beta1-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 beta1-AR is not responsible for the lack of beta1-AR spontaneous activation, although it has been known that the beta1-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 beta2-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.