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.
Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA.
Khalid
K
Chakir
Yang
Y
Xiang
Dongmei
D
Yang
Sheng-Jun
SJ
Zhang
Heping
H
Cheng
Brian K
BK
Kobilka
Rui-Ping
RP
Xiao
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D009200:Myocardial Contraction / Q000187:drug effects*; D009206:Myocardium / Q000378:metabolism*; D011412:Propanolamines / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions
D000319:Adrenergic beta-Antagonists; D007093:Imidazoles; D011412:Propanolamines; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C026777:ICI 118551; C049763:CGP 20712A; D000242:Cyclic AMP; D007545:Isoproterenol