Chakir Khalid, Xiang Yang, Yang Dongmei, Zhang Sheng-Jun, Cheng Heping, Kobilka Brian K, Xiao Rui-Ping
Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA.
Mol Pharmacol. 2003 Nov;64(5):1048-58. doi: 10.1124/mol.64.5.1048.
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.
众所周知，β2 - 肾上腺素能受体（β2-AR）表现出强大的非配体依赖性活性，而在密切相关的β1-AR亚型中，这种特性则弱得多。为了确定β2-AR中负责自发受体激活的潜在结构域，我们构建了三个嵌合体，其中β1-AR的第三个细胞内环（β1/β2-Li3）或羧基末端（β1/β2-CT）或这两个结构域（β1/β2-Li3CT）被β2-AR的相应部分所取代。利用腺病毒基因转移技术，我们在缺乏天然β1-AR和β2-AR的小鼠心肌细胞（β1/β2双敲除）中分别表达这些β1/β2-AR嵌合体，并检测它们可能的自发活性。与感染表达β-半乳糖苷酶的对照腺病毒或表达野生型β1-AR的腺病毒相比，这些β1/β2-AR嵌合体的过表达在无激动剂刺激的情况下显著提高了基础cAMP积累和心肌细胞收缩力。这些效应可被β2-AR反向激动剂（±）-1-[2,3-（二氢-7-甲基-1H-茚-4-基）氧基]-3-[(1-甲基乙基)氨基]-2-丁醇（ICI 118,551；5×10-7 M）完全逆转，无论百日咳毒素对Gi的抑制作用如何，但一组β1-AR拮抗剂，包括[2-（3-氨基甲酰基-4-羟基苯氧基）-乙基氨基]-3-[4-（1-甲基-4-三氟甲基-2-咪唑基）-苯氧基]-2-丙醇甲磺酸盐（CGP20712A）、倍他洛尔、比索洛尔和美托洛尔，均不能逆转这些效应。此外，我们已经表明，β1-AR的C末端突触后致密物95/盘状大蛋白/ZO-1（PDZ）基序与β1-AR缺乏自发激活无关，尽管已知β1-AR的PDZ基序可防止受体在心肌细胞中发生激动剂诱导的转运和Gi偶联。综上所述，目前的结果表明，第三个细胞内环和C末端均参与β2-AR的自发激活，并且这两个结构域中的任何一个似乎都足以赋予受体自发活性。
