The Third Intracellular Loop and the Carboxyl Terminus of β₂-Adrenergic Receptor Confer Spontaneous Activity of the Receptor

Abstract

It is well established that the ␤ 2 -adrenergic receptor (␤ 2 -AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related ␤ 1 -AR subtype. To identify the potential domain(s) of ␤ 2 -AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (␤ 1 /␤ 2-Li3) or the carboxyl terminus (␤ 1 /␤ 2-CT ) or both domains (␤ 1 /␤ 2-Li3CT ) of ␤ 1 -AR are replaced by the corresponding parts of the ␤ 2 -AR. Using adenoviral gene transfer, we individually expressed these ␤ 1 /␤ 2 -AR chimeras in mouse cardiomyocytes lacking both native ␤ 1 -AR and ␤ 2 -AR (␤ 1 /␤ 2 double knockout), and examined their possible spontaneous activities. Overexpression of these ␤ 1 /␤ 2 -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 ␤-galactosidase or an adenovirus expressing wild type ␤ 1 -AR. These effects were fully reversed by a, regardless of inhibition of G i with pertussis toxin, but not by a panel of, betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of ␤ 1 -AR is not responsible for the lack of ␤ 1 -AR spontaneous activation, although it has been known that the ␤ 1 -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 ␤ 2 -AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.␤-ARs are prototypical members of G protein-coupled receptor (GPCR) superfamily, which shares a common overall structure feature: the seven hydrophobic transmembrane helical domains, an extracellular N terminus, and an intracellular C terminus. Stimulation of ␤-ARs by catecholamines activates the classic Gs-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway, which, in turn, regulates multiple cellular processes, including metabolic regulation, muscle contraction, cell growth, and cell death (Xiao, 2001). In the heart, ␤-AR stimulation enhances the force and rate of myocardial contraction and relaxation in response to stress or exercise, allowing the heart to increase its output by severalfold within seconds.According to the ternary complex model and the cubic ternary complex model, GPCRs, including ␤-ARs, exist in an equilibrium between two functionally and conformationally distinct states: an inactive conformation (R) and an active conformation capable of activating G proteins (R*) (Samama et al., 1993;Bond et al., 1995;Neilan et al., 1999). In the absence of a receptor ligand, the receptor can undergo a spontaneous transition to the active state; the equilibrium between R and R* sets the level of basal receptor activation. Thus, an overexpression of a given receptor w...

Heterodimerization of β 1 - and β 2 -Adrenergic Receptor Subtypes Optimizes β-Adrenergic Modulation of Cardiac Contractility

“…These results are consistent with the previous notion that ␤ 2 AR, 16 -20 but not ␤ 1 AR, 16,21 exhibits spontaneous activity. Surprisingly, the Adv-␤ 2 AR infection-induced augmentation in the baseline contractility was fully prevented when cells were coinfected with Adv-␤ 1 AR (50 moi for each) (Figure 2A).…”

“…Because both ␤ 1 AR-and ␤ 2 AR-induced positive inotropic effects are mediated by a cAMP-dependent mechanism, [21][22][23] we next investigated the cAMP response to ␤AR stimulation in DKO cardiomyocytes expressing either or both ␤AR subtypes. Compared with that of uninfected WT or DKO cells, the baseline cAMP level was unchanged in myocytes expressing ␤ 1 AR, but augmented by 2.1-fold in cells infected with Adv-␤ 2 AR (100 moi) ( Figure 4A), caused by spontaneous ␤ 2 AR activity.…”

Sustained β 1 -Adrenergic Stimulation Modulates Cardiac Contractility by Ca 2+ /Calmodulin Kinase Signaling Pathway

“…12 cAMP level was measured with the cAMP ( 3 H) assay system (Amersham Biosciences) as described previously. 24 …”

“…␤ 1 AR radioligand binding studies were performed in membranes (25 to 100 g/tube) using the nonselective ␤AR antagonist ligand 125 I-cyanopindolol ( 125 I-CYP, 1 to 300 pmol/L) as described previously. 24 Nonspecific binding was determined in the presence of 10 mol/L propranolol. The maximal numbers of binding sites (B max ) and equilibrium dissociation constants (Kd) for 125 I-CYP were determined by Scatchard analysis.…”

An 80-Amino Acid Deletion in the Third Intracellular Loop of a Naturally Occurring Human Histamine H3 Isoform Confers Pharmacological Differences and Constitutive Activity

“…For other G-protein-coupled receptors (GPCRs), the IL3 has been shown to dictate G-protein specificity (Burstein et al, 1996;Senogles et al, 2004) and bind ␤-arrestins (Gelber et al, 1999) and calmodulin (Turner et al, 2004). Furthermore, the carboxyl terminus of the IL3 has been shown to play a role in constitutive activity of GPCRs (Chakir et al, 2003). In view of the relative high abundance of the hH 3 R(365) isoform and the known importance of the IL3 loop in GPCR-mediated signal transduction, we pharmacologically characterized the hH 3 R(445) and the hH 3 R(365) isoforms in full detail in this study.…”