The Third Intracellular Loop and the Carboxyl Terminus of  2 - Adrenergic Receptor Confer Spontaneous Activity of the Receptor KHALID CHAKIR, YANG XIANG, DONGMEI YANG, SHENG-JUN ZHANG, HEPING CHENG, BRIAN K. KOBILKA, and RUI-PING XIAO Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland (K.C., D.Y., S.-J.Z., H.C., R.-P.X.); and Department of Molecular and Cellular Physiology, Stanford University Medical Center, Stanford, California (Y.X., B.K.K.) Received April 4, 2003; accepted July 25, 2003 This article is available online at http://molpharm.aspetjournals.org 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 respon- sible 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 na- tive  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 accumula- tion and myocyte contractility in the absence of agonist stim- ulation 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  2 -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  1 -AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]- 3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2- propanolmethanesulfonate (CGP20712A), betaxolol, biso- prolol, 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 cardiomyo- cytes. 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 re- ceptor (GPCR) superfamily, which shares a common overall structure feature: the seven hydrophobic transmembrane he- lical domains, an extracellular N terminus, and an intracel- lular C terminus. Stimulation of -ARs by catecholamines activates the classic Gs-adenylyl cyclase-cAMP-protein ki- nase 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 sev- eralfold 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 would be ex- pected to proportionally increase the number of R* state receptors. K.C. and Y.X. contributed equally to this study. ABBREVIATIONS: -AR, -adrenergic receptor; GPCR, G protein-coupled receptor; PKA, protein kinase A; DKO, double knockout; WT, wild type; ICI 118,551, ()-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; CGP20712A, [2-(3-carbamoyl-4-hydroxyphe- noxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate; m.o.i., multiplicity of infection; MEM, min- imal essential medium; FBS, fetal bovine serum; ICYP, iodocyanopindolol; PBS, phosphate-buffered saline; -gal, -galactosidase; ISO, isoproterenol; PTX, pertussis toxin; Adv, adenovirus. 0026-895X/03/6405-1048 –1058 MOLECULAR PHARMACOLOGY Vol. 64, No. 5 U.S. Government work not protected by U.S. copyright 2566/1101526 Mol Pharmacol 64:1048–1058, 2003 Printed in U.S.A. 1048 at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from